TW202034948A - Combination vaccine composition comprising reduced dose inactivated poliovirus and method for preparing the same - Google Patents

Abstract

The present disclosure relates to a fully liquid immunogenic composition comprising a combination of antigens/immunogens. The immunogenic composition comprises optimum amount of antigens/immunogens to confer protection against a number of diseases. The composition exhibits improved immunogenicity and stability. A process for preparing the vaccine composition is also disclosed.

Description

Combined vaccine composition containing reduced dose of inactivated polio virus and preparation method thereof

The present invention relates to the field of biotechnology, and more specifically, the present invention relates to a method for preparing a multi-dose combination vaccine composition comprising a set of antigens/immunogens and preservatives. The invention further relates to an improved method in the field of combined vaccine manufacturing.

Combination vaccines that can provide immunogenicity against multiple diseases are always more advantageous than monovalent vaccines because they reduce the number of injections, reduce the complications associated with multiple intramuscular injections, reduce management and production costs, and reduce Inventory costs, reduce the risk of delayed or missed vaccines, and improve patient compliance by reducing the number of individual vaccinations. Moreover, compared with the combined vaccine that requires rehydration, the full liquid formulation of the combined vaccine has obvious advantages. It is found that the average preparation time of a full liquid vaccine is almost half of that of a non-full liquid vaccine. Almost all medical staff (97.6%) said that they prefer to use whole liquid vaccines in daily practice. (Reference: Soubeyrand B, et al.; Assessment of preparation time with fully-liquid versus non-fully liquid paediatric hexavalent vaccines. A time and motion study; Vaccine 2015; 33:3976-82).

The currently known and available combination vaccines may not contain the appropriate preparation of the appropriate antigen in the appropriate immunogenic form to achieve the required level of safety, efficacy and immunogenicity in susceptible populations against multiple diseases in a single injection . The number of different vaccine combinations that can be produced with only a few other antigens is considerable. By adding 1 to 4 other antigen components (such as HIB (lyophilized or liquid), HBV, IPV, HAV) to DTwP or DTaP, 44 possible different vaccine combinations can be generated. If you consider individual components from different manufacturers, the number will increase to thousands. Since each individual new combination vaccine must be developed separately (considering the difference of the components according to the source) to prove safety, stability, compatibility and efficacy, the development of all these vaccines has become a difficult task. Combination vaccine antigen: diphtheria and tetanus antigen

Diphtheria and tetanus are acute infections caused by Corynebacterium diphtheriae and Clostridium tetani, respectively. In both cases, the effective exotoxins of these bacteria are the cause of clinical disease. Vaccines that provide protection against these bacteria contain toxins that are chemically modified to form toxoids, a chemically modified toxin that is no longer toxic but still antigenic. Diphtheria and tetanus toxin are produced by growing Corynebacterium diphtheriae and Clostridium tetani in a medium containing bovine extract. Use the following treatments to inactivate toxins, including heating, UV, formalin/formaldehyde, glutaraldehyde, acetylethyleneimine, etc., to prepare toxoids [diphtheria toxoid (D) and tetanus toxoid (T)]. Concerns about bovine spongiform encephalopathy (BSE), infectious spongiform encephalopathy (TSE), and Creutzfeldt-Jakob disease (CJD and variant CJD disease) may stem from the animal components used in the growth medium containing the vaccine Spread the cow extract. (Reference: WHO Guidelines on Transmissible Spongiform Encephalopathies in relation to Biological and Pharmaceutical Products; 2003 &EMEA/CPMP/BWP/819/01; April 24, 2001). Pertussis antigen

In the 1940s, a whole-cell vaccine containing chemically inactivated and heat-inactivated Bordetella pertussis was introduced, which can greatly reduce the incidence of whooping cough caused by Bordetella pertussis.

Whole-cell DTP vaccines are usually associated with several local adverse events (such as erythema, swelling and pain at the injection site), fever, and other mild systemic events (such as lethargy, irritability, and anorexia) (reference: Cody CL, Baraff LJ, Cherry JD, Marcy SM, Manclarck CR; The nature and rate of adverse reactions associated with DTP and DT immunization in infants and children. Paediatrics 1981; 68:650-60) and (reference: Long SS, DeForest A, Pennridge Pediatric Associates, Longitudinal study of adverse reactions following Diphtheria-tetanus-pertussis vaccine in infancy. Paediatrics 1990; 85:294-302). Among children receiving whole-cell DTP vaccines, more serious systemic events (for example, seizures {with or without fever} and hypotonic and hyporesponsive episodes) occurred less frequently (1 in 1,750 administrations) Proportion) (Reference: Cody CL, Baraff LJ, Cherry JD, Marcy SM, Manclarck CR; The nature and rate of adverse reactions associated with DTP and DT immunization in infants and children. Paediatrics 1981; 68:650-60). The incidence of acute encephalopathy is even lower (ratio of 0-10.5 cases in one million administrations). Experts believe that the whole-cell pertussis vaccine can cause long-lasting brain damage in some rare cases. (Reference: Institute of Medicine; DPT vaccine and chronic nervous system dysfunction, a new analysis; Washington D.C., National Academy Press, 1994).

Several reports have cited the relationship between whole-cell pertussis vaccination, reactogenicity, and severe side effects, which led to a decline in vaccine acceptance and thus a re-epidemic (Miller, DL, Ross, EM, Alderslade, R., Bellman, MH, and Brawson, NSB (1981). Pertussis immunization and serious acute neurological illness in children: Brit Med. J. 282: 1595-1599).

Whole-cell pertussis (wP)-related adverse reactions hinder its continued use on a global scale. Therefore, in the industrialized world, WP-based combination vaccines are gradually being replaced by acellular pertussis-based combination vaccines.

Recently, a pertussis vaccine of defined composition has been developed. It has been previously reported that all liquids are based on hexavalent acellular pertussis vaccine (DTaP IPV PRP-T-HBsAg) (EP1028750).

Infanrix® Hexa (GSK) is currently the only hexavalent pediatric vaccine containing Salk IPV sold in the world. The product (DTaP3 -IPV-HBV//Hib) is sold as a prefilled syringe of the pentavalent product. The pentavalent product and the freeze-dried Hib antigen PRP-T conjugate are packaged in a separate vial, and the rest is used before use Vaccine rehydration.

The second hexavalent vaccine Hexyon® (also known as Hexacima® and Hexaxim®) is a full liquid hexavalent vaccine from Sanofi Pasteur; however, it also has aP. The vaccine may target European and global private markets.

The seven-valent combination vaccine is being developed by Bharat Biotech International, which contains DT, acellular pertussis, Sabin IPV (type I: 40 DU, type 2: 8 DU, type 3: 32DU), single-strain inactivated rotavirus (G9 strain) , Namely 116E strain), Haemophilus influenzae type B PRP conjugate with TT and recombinant hepatitis B vaccine.

However, there is growing concern about the long-term effectiveness of acellular pertussis (aP) vaccines, especially in developing countries. Recent reports indicate that adolescence's immunity to whooping cough is reduced, which is the reason for the increase in cases of infants younger than six months before they are fully vaccinated. It is estimated that the effectiveness of the vaccine is 24% in children 8 to 12 years of age immunized with aP in infancy. An observational study conducted in Australia also showed that the prevalence of aP-vaccinated adolescents in infancy is higher than that of wP-vaccinated adolescents (relative risk is 3.3, 95% confidence interval 2.4-4.5).

From a cost perspective, due to manufacturing differences and licensing costs, the cost of aP antigens has historically been 10 to 30 times higher than that of wP antigens, thus posing an economic burden on developing countries. Therefore, the cost of the hexavalent vaccine based on wP will be more suitable for use in the public sector in resource-poor countries.

Therefore, due to cost and concerns about the long-term effectiveness of aP vaccine, especially in developing countries, the use of whole-cell pertussis (wP) in the hexavalent vaccine used in developing countries has become important.

Compared with the best whole-cell pertussis (wP) vaccine, the aP vaccine is less effective in mass immunization programs (Vickers et al. 2006; Cherry 2012).

Recent outbreak studies in highly immunized populations have shown that the protection time of aP vaccine is too short (Klein et al. 2012; Misegades et al. 2012), resulting in a decline in the immunity of older children and adolescents, and cases in this age group correspond Increase (Skowronski et al. 2002; Klein et al. 2012). This is in contrast to the wP vaccine, which can provide good protection during adolescence (Klein et al. 2012). Because of these shortcomings, in the countries that switched to aP vaccines in the 1990s, there is now a generation of children that are not only less able to resist pertussis, but also have a weaker response to booster immunization. This is because the child’s vaccination may determine its impact on the future Boost the immune response of vaccination (Podda et al. 1995; Mascart et al. 2007; Sheridan et al. 2012; Liko, Robison and Cieslak 2013; Smits et al. 2013).

One of the most important factors leading to the reactogenicity of wP is the presence of lipo-oligosaccharides (LOS), which are endotoxins derived from the outer membrane of bacteria.

The inactivation of toxins in the wP vaccine can be accomplished by various methods, but no active thermolabile toxins should be detected in the final product. The whole-cell pertussis (wP) method implemented by many manufacturers to inactivate wP toxin uses heat treatment/formalin. Some reports cited the use of thimerosal to inactivate wP. However, the use of thimerosal can cause loss of antigenicity of IPV (Vaccine 1994 Vol. 12 No. 9 851-856. Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine). Therefore, in the case of a combination vaccine containing IPV, it may be It needs to be placed in a vial separate from the wP containing thimerosal to maintain its effectiveness over time or to change the overall inactivation of pertussis source. Some antigens, active PT, can also be used as immune response modifiers, and significant differences in immune responses to various antigens have been observed between different vaccines (WHO, 1993).

According to the use of in vitro or in vivo tests, the chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a dramatic decrease in endotoxin-related toxicity (up to 97%). LOS extraction does not affect the integrity of the product, and more importantly, does not affect the effectiveness and/or stability of DTP. Moreover, almost no differences in antibody and T cell responses were observed. (Reference: Waldely Oliveira Dias et al.; An improved whole cell pertussis vaccine with reduced content of endotoxin; Human Vaccines & Immunotherapeutics 9:2, 339–348; February 2012). Hepatitis B antigen

There are many strains of hepatitis virus. Hepatitis B is a disease caused by the hepatitis B virus (HepB), which infects the liver of humans and causes inflammation called hepatitis. The vaccine against the disease contains a viral envelope protein, the hepatitis B surface antigen (HBsAg). Vaccines that have been used for mass immunization are now available, such as Merck's products RecombivaxHB® and Comvax®, Glaxo SmithKline Biologicals' Engerix-B® and Pediarix®. Compared with the HepB single antigen vaccine, the combined vaccine with hepatitis B component has a higher completion rate and compliance results. (See: Kurosky et al.; Effect of Combination Vaccines on Hepatitis B Vaccine Compliance in Children in the United States; The Pediatric Infectious Disease Journal. 36(7):e189–e196, July 2017). Some references cited the adsorption of the combination of hepatitis B surface antigen and other antigens on aluminum phosphate. The Hexavac® combination vaccine was withdrawn from the market due to the low immunogenicity of the hepatitis B component. Therefore, a combination vaccine composition containing hepatitis B antigen with sufficient or enhanced immunogenicity is needed. Haemophilus influenzae (Hib) antigen

Haemophilus influenzae is a gram-negative coccus that is a normal part of the upper respiratory tract flora. Haemophilus influenzae type b (Hib b) is the main cause of invasive blood-borne infections in children with meningitis, and it is also the main cause of meningitis in the first 2 years of life. In 1987, Canada began to use polysaccharide vaccine [phosphopolyribosyl ribitol (PRP)] to immunize Haemophilus influenzae. Hib's phosphate polyribosyl ribitol (PRP) capsule is the main virulence factor of organisms. The main contributor to the anti-PRP anti-system serum bactericidal activity, the increase in antibody level is related to the decrease in the risk of invasive diseases. PRP is a T-cell-independent antigen and therefore has the following characteristics: a) Induces poor antibody responses in infants and children under 18 months of age, b) Changes in antibody responses compared with T-cell-dependent antigens And the amount is smaller; c) produces a higher proportion of immunoglobulin M (IgM), and d) fails to induce a booster response.

Initially, vaccines based only on PRP components proved ineffective for infants. Further efforts are directed to PRP conjugate vaccines, in which PRP is conjugated with a protein called carrier protein (such as Neisseria meningitidis, diphtheria toxoid, tetanus toxoid, and CRM 197 outer membrane protein). The inclusion of the Hib conjugate component in the combination vaccine is associated with reduced Hib immunogenicity. In addition, Hib-conjugates are unstable in aqueous media and cannot be stored in this form for a long time. Therefore, the PRP polysaccharide of Haemophilus influenzae b (Hib) is usually formulated as a dry solid, which is rehydrated with liquid preparations of other antigens during delivery. For example in Infanrix® hexa (WO99/48525). Polio antigen

Different types of vaccines can be used: • Live attenuated (weak) oral polio vaccine (OPV) developed by Dr. Albert Sabin in 1961. OPV containing Sabin strain was administered orally. • Inactivated (dead) polio vaccine (IPV) developed by Dr. Jonas Salk in 1955. IPV containing the Salk strain was administered as an injection. • Recently, Sabin inactivated poliovirus for injection has been developed, which is prepared by inactivating Salk strain poliovirus with formalin, and is also available in commercial products.

Both live attenuated (OPV) and inactivated (IPV) polio vaccines have effectively controlled polio diseases worldwide. The polio vaccine may comprise Salk or Sabin strains.

In 1955, Dr. Jonas Salk successfully inactivated wild-type poliovirus, making it an injectable preparation, and named it the Salk strain. This Salk strain includes Mahoney 1, MEF 2, and Saukett 3. Etc. have been used for polio vaccine. Sabin strains include Sabin1 and Sabin2 strains.

The currently accepted standard dose of polio vaccine contains 40 D antigen unit of inactivated poliovirus type 1 (Mahoney), 8 D antigen unit of inactivated poliovirus type 2 (MEF-1) and 32 D The antigen unit type 3 inactivates poliovirus (Saukett), such as Infanrix-hexa® (WO99/48525).

IPV is currently available as a non-adjuvant independent preparation, or in a variety of combinations, including DT-IPV (used with diphtheria and tetanus toxoid) and hexavalent IPV vaccine (with whooping cough, hepatitis B, influenza B hematocrit) Bacillus and adjuvant together), such as Infanrix® hexa (WO99/48525).

However, compared with OPV, the total production cost of IPV is much higher. This is mainly due to the need for: (i) more viruses per dose; (ii) additional downstream processing (ie concentration, purification and inactivation); and related QC testing (iii) downstream antigen loss or low recovery Rate and iv) capacity. So far, in low- and middle-income countries, financial challenges have been the main drawback of IPV innovation and implementation.

After the eradication of poliovirus, the global demand for IPV may increase from the current 80 million doses to 450 million doses each year. Therefore, it may be necessary to "extend" the IPV supply.

The applicant unexpectedly found that compared with the standard dose of IPV antigen, the reduced dose of IPV showed non-inferiority/equivalent resistance to polio. When the supply of conventional vaccines is insufficient to meet global demand, or when the production cost of conventional vaccines prevents the vaccines from being sold at affordable prices in developing countries, a lower dose of IPV antigen is required to provide anti-infection A low-dose effective vaccine preparation with protective effect. It also needs to be exposed to a lower dose of IPV; it may be safer than the existing commercially available formulations. Therefore, various strategies for providing IPV at a more affordable price need to be evaluated. Therefore, a combination vaccine containing a reduced dose of IPV can make it cheaper and easier to administer.

In the case of pandemic influenza vaccines, the use of adjuvants can reduce the dose, increase availability and reduce vaccine costs. Therefore, it has been speculated that adjuvant vaccine formulations for IPV will reduce costs and will also increase the number of IPV doses available worldwide.

In addition, aluminum salts have been considered safe, have been used in combination vaccines containing IPV, have the lowest development barriers and are cheap to manufacture. However, it is known that aluminum adjuvants can significantly reduce the dose.

In addition, the whole cell pertussis antigen present in the hexavalent vaccine has proven to be a powerful immunostimulant. Due to the immunostimulatory effects of aluminum phosphate adjuvant and whole-cell pertussis vaccine, we speculate that a good immune response can be obtained when the IPV dose is reduced. Other antigens

Other antigens that may be included in the combined vaccine are Haemophilus influenzae (serotypes a, c, d, e, f and unencapsulated strains), hepatitis (A, C, D, E, F, and G strains), Neisseria meningitidis A, B, C, W, X, Y, influenza, pneumococcus, streptococcus, anthrax, dengue fever, malaria, measles, mumps, rubella, BCG, Japanese encephalitis, rotavirus, smallpox , Yellow fever, typhoid fever, shingles, varicella virus, etc.

The range and types of antigens used in the combination vaccine depend on the age of the target group to be used, such as infants, toddlers, children, adolescents and adults. It can prevent the earliest known infections of Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, and optionally inactivated poliovirus (IPV) and/or hepatitis B virus and/or Haemophilus influenzae type B Combination vaccines are known (see eg WO 93/24148, WO97/00697, WO2000/030678, WO2008/028956, US 6013264 and WO2005089794).

However, the well-proven phenomenon of antigen competition has complicated and hindered the development of multivalent vaccines. This phenomenon refers to the observation that compared with the immune response to these antigens when administered alone, the administration of multiple antigens together usually results in a weakened response to certain antigens.

At the same time, multi-dose vaccines should contain preservatives to avoid contamination by harmful microorganisms. For vaccine products exported to underdeveloped countries, considering the environment and distribution method of the country where the vaccine is to be used, multi-dose vaccines containing preservatives are better. Examples of preservatives that have been used include Phemerol Chloride (Phemerol), Thiomersal, Phenol, Formaldehyde, and 2-Phenoxyethanol (2-PE) known in the art. Preservatives suitable for vaccines should be environmentally safe, effective against bacteria, yeast and other fungi, and have no negative impact on the immunogenicity of the vaccine.

Thiomersal is a derivative of ethyl mercury and has been widely used as a preservative in many vaccines. It is known that thimerosal can prevent the growth of contaminating microorganisms and maintain a sterile state during the storage or use of vaccine products. Many combination vaccines that have obtained the WHO prequalification (PQ) contain thimerosal as a preservative. However, there are reports related to some allergic reactions to thimerosal (about 16% of the population), mainly in the form of delayed local hypersensitivity, including redness and swelling at the injection site.

Furthermore, inactivated polio vaccines conventionally use 2-PE instead of thimerosal as a preservative, because it is known that thimerosal is used as a preservative in inactivated polio vaccines, even if stored in the refrigerator, the vaccine will be used within a week The effectiveness is reduced by 50% or more. (Vaccine 1994 Vol. 12 No. 9 851-856. Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine).

Combination vaccines (including D, T, wP, Hib, HBsAg and IPV) also use 2-PE (WO2010046934, WO2008020322 and WO2012093406) at a concentration of 5 mg/mL.

However, at 2-8°C, in the combination vaccine based on DPT, 2-PE was found to have weaker antimicrobial activity against yeast and fungi than thimerosal. It is an option to increase the preservative efficacy of the combined vaccine by increasing the amount of 2-PE to meet the required standards. However, increasing the concentration of 2PE may cause safety issues in young children among subjects receiving the combination vaccine, leading to regulatory obstacles to the approval of such vaccines.

Therefore, it would be advantageous to improve the preservative efficacy of the combined vaccine by combining 2-PE with at least one other preservative that meets safety and regulatory standards. Examples of preservatives other than 2-PE that can be used include chlorhexidine (Phemerol), parabens, phenol, and formaldehyde, which are known in the art.

According to USP, BP and EP, it was found that methyl paraben, propyl paraben and benzyl alcohol passed the antimicrobial test. In addition, these preservatives are non-toxic but effective. The toxicity of parabens is relatively low due to the convenience and rapidity of the human body to get rid of these drugs. The LD50 of methylparaben in mouse peritoneum is 1g/kg. It has not been found that a mixture of methyl paraben and propyl paraben can be used in commercial vaccines.

The applicant found that the preservative effect of a mixture of 2-phenoxyethanol and parabens (for example, methyl paraben, propyl paraben) is more effective than 2-phenoxyethanol alone.

Furthermore, the applicant found that the immunogenicity, reactogenicity, stability and maintenance of the correct form of the antigen in the combination vaccine composition depend on the formulation of the composition, which includes: a) the method of making a single antigen b) Order of antigen addition c) For certain antigens, use a specific amount of a specific adjuvant, d) Adsorption of antigen on the adjuvant alone or combined adsorption, where combined adsorption has its advantages in an easy-to-operate form, and the disadvantages include the first The pre-adsorbed antigen may be partially or completely desorbed during the addition of subsequent antigens. Since the previous antigen may be saturated with adsorption capacity, the antigen added in the last step may not be completely adsorbed. Weakly adsorbed antigens may desorb during storage. e) The degree of adsorption of the antigen on the adjuvant f) Use the lowest alum concentration g) Use the optimal concentration and type of preservative h) Use various parameters, including stirring, temperature and pH. purpose

Some of the objectives of the present invention fulfilled by at least one embodiment in the text are as follows:

An object of the present invention is to improve one or more of the problems in the prior art or at least provide a useful alternative.

Another object of the present invention is to provide a full liquid combination vaccine suitable for preventing and preventing infections caused by diphtheria, tetanus, pertussis, polio, Haemophilus influenzae and hepatitis B, or preventing, improving or delaying them Onset or progression of clinical manifestations.

Yet another object of the present invention is to provide a whole liquid combination vaccine containing various reduced doses of inactivated poliovirus (IPV) antigens. Compared with the standard doses of IPV antigens, the vaccine shows a non-toxic effect on polio. Inferiority/equivalent resistance.

Yet another object of the present invention is to provide a complete liquid containing at least one paraben, namely methyl paraben or propyl paraben preservative and 2-phenoxyethanol (2-PE) Combined vaccine to improve the antiseptic effect of multiple dose combined vaccine.

Yet another object of the present invention is to provide an improved method for manufacturing such a combination vaccine composition/preparation, wherein the vaccine shows improved immunogenicity, reduced reactogenicity, improved stability and further satisfies each The criteria for seroprotection of the immunogenic components.

From the following description, other objects and advantages of the present invention will become more apparent, and are not intended to limit the scope of the present invention.

A combination vaccine composition comprising a reduced dose of inactivated poliovirus (IPV) antigen and other antigens/immunogens and at least one paraben (ie methyl paraben or paraben) as a preservative Propyl hydroxybenzoate) and 2-phenoxyethanol (2-PE) combination, which improved the antiseptic efficacy of the multi-dose combination vaccine and its preparation method.

The present invention relates to a combined vaccine composition, which comprises the following: a) High-purity diphtheria toxoid (D) and tetanus toxoid (T) produced using semi-synthetic medium, and then detoxified and adsorbed on aluminum phosphate adjuvant respectively to improve immunogenicity b) Inactivated whole-cell Bordetella pertussis (wP) component prepared using a combination of heat and chemical inactivation. Specific Bordetella pertussis strains have a specific ratio, resulting in reduced reactogenicity and improved efficacy. c) Haemophilus influenzae type b (Hib) capsular polysaccharide antigen (PRP) conjugated with carrier protein (CP) d) Reduced-dose Salk or Sabin (inactivated poliovirus) IPV, which shows an effect equivalent to the standard dose prepared by using a modified formaldehyde inactivation method and optionally adsorbed to aluminum phosphate adjuvant . e) Hepatitis B (HepB) surface antigen that is adsorbed alone on aluminum phosphate adjuvants resulting in enhanced immunogenicity f) Minimum alum content to ensure reduced reactogenicity g) At least one paraben (ie methyl paraben or propyl paraben) other than 2-phenoxyethanol (2-PE) is used as a preservative.

Although the present invention may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion. It should be understood that the present invention can be considered as an example of the principles of the present invention and is not intended to limit the scope of the present invention to The contents explained and disclosed in this manual. The embodiments are provided in order to fully and fully convey the scope of the present invention to those skilled in the art. Many details related to specific components and methods are explained to provide a complete understanding of the embodiments of the present invention. It will be obvious to those skilled in the art that the details provided in the embodiments should not be construed as limiting the scope of the present invention. In some embodiments, the known composition, the known method and the known technique are not described in detail.

In the present invention, the terms used are only for the purpose of explaining specific embodiments, and such terms should not be considered as limiting the scope of the present invention. As used in the present invention, unless the context clearly indicates otherwise, the forms "a (a)/(an)" and "the" are also intended to include plural forms.

The terms first, second, third, etc. should not be construed as limiting the scope of the present invention, because the aforementioned terms can only be used to distinguish one element, component, region, layer or section from another component, region, layer or section. Unless the present invention clearly proposes, terms such as first, second, third, etc. used herein do not imply a specific order or order. The invention provides an immunogenic composition and a preparation method thereof.

The term "vaccine"" may be replaced by the term "immunogenic composition" as appropriate, and vice versa.

"D antigen unit" (also known as "international unit" or IU): The D antigen form of polio virus induces protective neutralizing antibodies. The D antigen unit referred to herein (for example, in the vaccine of the present invention) is added to each human dose of the formulated vaccine (usually 0.5 mL final volume) before preparing the final vaccine. The total D antigen unit measured. A reliable method for measuring D-antigen units is well known in the art and is disclosed, for example, by the European Pharmacopoeia. For example, the ELISA test described in Example 1 below ("D antigen quantification by ELISA") can be used to measure D antigen units. The European Pharmacopoeia provides test samples (European Pharmacopoeia Biological Reference Preparations, available from the European Pharmacopoeia Secretariat, for example code P 216 0000) to standardize such methods among manufacturers (Pharmeuropa Special Issue, Bio 96-2). Therefore, the D antigen unit value is well known in the art.

The term "dose" as used herein generally refers to one administration of the vaccine of the present invention, usually one injection. The typical human dose is 0.5 mL. Of course, various doses can be administered in the vaccine administration schedule.

The term "IPV" or the immunogenic composition containing these components herein is intended to mean inactivated poliovirus type 1 (for example Mahoney, preferably used), type 2 (for example MEF-1) or type 3 ( For example Saukett), or a combination of any two or all three Sabin serotypes 1, 2, and 3. For the purpose of the present invention, the full dose (or standard dose) (respectively based on 40-8-32 D antigenic units of Salk type 1, 2 and 3 IPV) IPV immunogenic composition can be Polyiovac® ( Serum Institute of India Pvt. Ltd.). Therefore, it is pointed out in this article that compared with the standard dose of Salk-based IPV, the immunogenic composition of the present invention reduces (decreases) one, two, or three times the dose, which means that in each dose of the vaccine The formulated D-antigen unit is equal to the X% reduction in the dose of 40, 8 and/or 32 D antigen units of type 1, 2 and/or 3 IPV respectively (as measured in each bulk IPV antigen type).

Throughout the specification, the term "sugar" may mean polysaccharides or oligosaccharides, and includes both. Capsular saccharide antigens can be full-length polysaccharides, or can be extended to bacterial "large and small sugars" and "oligosaccharides" (which naturally have a small number of repeating units, or they are polysaccharides whose size is reduced for ease of handling, but can still be in the host Inducing a protective immune response.

According to the first embodiment of the present invention, the combination vaccine composition comprises an antigen/immunogen group selected from but not limited to: diphtheria toxoid (D), tetanus toxoid (T), whole cell Bordetella pertussis (wP) ), Haemophilus influenzae type b (Hib) PRP-CP conjugate, hepatitis B (HepB), reduced-dose inactivated poliovirus (IPV), and further comprising 2-phenoxyethanol and at least one pair A combination of paraben preservatives.

According to the second embodiment of the present invention, the combination vaccine composition may further comprise one or more antigens each selected from the group consisting of but not limited to: Haemophilus influenzae (a, c, d, e, f serotype And unencapsulated strains), hepatitis (A, C, D, E, F and G strains), Neisseria meningitidis A, B, C, Y, W-135 or X, influenza, Staphylococcus aureus , Salmonella typhi antigen, acellular pertussis antigen, modified adenylate cyclase, malaria antigen (RTS, S), pneumococcus, streptococcus, anthrax, dengue fever, malaria, measles, mumps, rubella, BCG, human Papilloma virus, Japanese encephalitis, dengue fever, Zika, Ebola, Qu Gong, rotavirus, smallpox, yellow fever, flavivirus, herpes zoster, varicella virus antigens.

According to a third embodiment of the present invention, the IPV strain used in the combination vaccine composition includes an inactivated Sabin strain selected from the group of type 1, type 2 and type 3 or selected from Mahoney type 1, MEF type 2 and type 3 The inactivated Salk strain of the Saukett group.

In one aspect of the third embodiment, the polio virus can be grown by the following methods: • CCL81-VERO (monkey kidney) cells are used as host cells for the growth of poliovirus (ie Sabin and Salk strains). • After infecting the host cells with the desired polio strain and incubating for 72 hours, pool the media containing the virus and cell debris and collect them in a single container. • Use a 100 KDa filter cartridge to filter the filtrate in tangential flow; diafiltration with phosphate buffer and purify by anion exchange chromatography. • Before administering to patients, the virus must be inactivated using appropriate inactivation methods.

However, the inventors unexpectedly discovered that the high percentage loss of D-antigen after formaldehyde inactivation may be due to the presence of phosphate buffer, which unexpectedly caused undesired aggregation of poliovirus particles.

Therefore, an important aspect of the present invention includes an improved method of formalin inactivation, which includes the following steps: a) The purified virus pool is prepared from phosphate buffered saline in a pH range of 7 to 7.5 (30 to 50 mM) Buffer exchange into Tris buffer, b) Add M-199 medium containing glycine (5 gm/l) to the above mixture c) Add 0.025% formaldehyde, then mix, d) Then incubate the mixture at 37°C for 5 to 13 At the same time, the virus mass was continuously stirred on a magnetic stirrer at the same time. e) On the 7th day, the incubation mixture was placed in the intermediate TFF system (100 KDa, 0.1 m ² ), and the final filtration was performed after inactivation. f) The subsequent filtration The subsequent materials are stored at 2-8°C, g) D-Ag ELISA is used for D-Ag unit determination

According to the fourth embodiment of the present invention, the IPV strain used in the combination vaccine composition includes an inactivated Sabin strain selected from the group of type 1, type 2 and type 3 with a reduced dose or selected from Mahoney type 1 and MEF type 2 And the inactivated Salk strain of the Saukett group type 3.

According to the fifth embodiment of the present invention, IPV (Sabin or Salk strain) can be added to the final combined vaccine composition without being adsorbed on any adjuvant alone.

According to one of the preferred aspects of the fifth embodiment, IPV (Sabin or Salk strain) can be adsorbed on the adjuvant, preferably in the aluminum phosphate or aluminum hydroxide salt of the combined vaccine, wherein the IPV antigen is against type 1 The adsorption percentage of IPV can be in the range of 10%-30%, type 2 IPV can be in the range of 60%-100%, and type 3 IPV can be in the range of 0%-25%.

According to the sixth embodiment of the present invention, one or more IPV (Sabin or Salk strain) components can be individually adsorbed on an adjuvant selected from the following group: aluminum salt (Al ³ +), such as aluminum hydroxide (Al (OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water emulsion, or a combination thereof. (For example before or after mixing with other components (if present)). If adsorbed, one or more IPV components can be adsorbed on aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate alone or together as a mixture.

IPV (Sabin or Salk strain) components can be adsorbed on aluminum salt by the following steps: • Take the required volume of autoclaved Al(PO) ₄ or Al(OH) ₃ to obtain the final result in a 50 ml container The concentration of alum (Al ³ +) is between 0.1 and 0.8 mg/dose. • Add IPV block with adjusted D-Ag unit and make up the volume with diluent (10x M-199 + 0.5% glycine). • Adjust the final The pH of the formulation and the final formulation with a pH between 6 and 7.5 is obtained.

In one aspect of the sixth embodiment, the adsorption of formalin-inactivated IPV can be selected at a concentration of 0.1 mg/dose, 0.2 mg/dose, 0.3 mg/dose, 0.4 mg/dose, 0.5 mg for each serotype. /Dose, 0.6 mg/dose, 0.7 mg/dose and 0.8 mg/dose, preferably 0.1 mg/dose to 1.25 mg/dose of alum (Al ³ +), and selected from 6.2, 6.3, 6.4, 6.5 , 6.6, 6.7 and 6.8, preferably at a pH of 6.5.

In yet another aspect of the sixth embodiment, the recovery percentage of D-antigen after formalin inactivation in the presence of Tris can be 50%, 60%, 70% or 80%, and the adsorption percentage after aluminum phosphate adsorption It can be between 70% to 80%, 80% to 90%, 90% to 99%, or 95% to 99%.

According to the seventh embodiment of the present invention, diphtheria toxin (exotoxin) and tetanus toxin (exotoxin) are obtained from Corynebacterium diphtheriae and Clostridium tetani, respectively, and then detoxified by an appropriate inactivation method. The diphtheria toxoid (D) and tetanus toxoid (T) thus obtained can be purified by gel filtration chromatography. The purified DT thus obtained is further used to formulate a combination vaccine.

In one aspect of the seventh embodiment, diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semi-synthetic medium composed of the following components in any combination of the following at optimal concentrations: Union 1:

Casein hydrolysate, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, β-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride Hydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate, ferrous sulfate and WFI. Union 2:

Casein hydrolysate, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, ß-alanine, pimelic acid, niacin, manganese chloride, L-cystine, calcium chloride dihydrate, orthophosphate dihydrate Potassium hydrogen, dipotassium hydrogen phosphate, ferrous sulfate and WFI. Union 3:

Casein hydrolysate, maltose monohydrate, glacial acetic acid, sodium lactate, ß-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride dihydrate, Potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate and WFI. Union 4:

Yeast extract, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, ß-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride dihydrate Materials, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate, ferrous sulfate and WFI.

According to the second aspect of the seventh embodiment, tetanus toxin is produced by growing Clostridium tetani in a semi-synthetic medium composed of the following components in an optimal concentration in any combination of the following: Union 1:

Casein digestive juice, calcium chloride, dipotassium hydrogen phosphate, anhydrous glucose, sodium chloride, magnesium sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, Ferric chloride, vitamin B12 solution, biotin, concentrated HCl, NaOH, absolute ethanol and WFI Union 2:

Casein digestive juice, calcium chloride, β-alanine, dipotassium hydrogen phosphate, anhydrous glucose, sodium chloride, magnesium sulfate, ferrous sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate , Niacin, L-cystine, ferric chloride, vitamin B12 solution, biotin, concentrated HCl, NaOH, absolute ethanol and WFI Union 3:

Casein digestive juice, calcium chloride, dipotassium hydrogen phosphate, anhydrous glucose, sodium chloride, zinc sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, Ferric chloride, vitamin B12 solution, biotin, concentrated HCl, NaOH, absolute ethanol and WFI Union 4:

Casein hydrolysate, calcium chloride, dipotassium hydrogen phosphate, anhydrous glucose, sodium chloride, magnesium sulfate, manganese chloride, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L- Cystine, ferric chloride, vitamin B12 solution, biotin, concentrated HCl, NaOH, absolute ethanol and WFI

In yet another aspect of the seventh embodiment, one or a combination of the following inactivation methods is used to detoxify diphtheria and tetanus toxins. The inactivation method includes heating, UV, formalin/formaldehyde, acetylethylene Amine etc.

According to the eighth embodiment of the present invention, the hepatitis (Hep) antigen used in the combination vaccine composition includes hepatitis antigen (HBsAg) derived from the surface of the hepatitis B strain.

In one aspect of the ninth embodiment, HBsAg can be prepared by one of the following methods: • By purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, because a large amount of HBsAg is synthesized in the liver and released into the bloodstream during HBV infection • Express protein by recombinant DNA method

According to the ninth embodiment of the present invention, diphtheria toxoid (D), tetanus toxoid (T), and hepatitis B surface antigen (HBsAg) are individually adsorbed on an adjuvant selected from the group consisting of aluminum salt (Al ^{3 +} ), such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposome or Oil-in-water emulsion or a combination thereof.

Still preferably, diphtheria toxoid (D), tetanus toxoid (T) and hepatitis B surface antigen (HBsAg) are individually adsorbed on aluminum phosphate.

In one aspect of the ninth embodiment, the diphtheria toxoid (D) antigen adsorbed on aluminum phosphate has an adsorption percentage of at least 50%.

In another aspect of the ninth embodiment, the tetanus toxoid (T) antigen adsorbed on aluminum phosphate has an adsorption percentage of at least 40%.

In yet another aspect of the ninth embodiment, the hepatitis B surface antigen (HBsAg) adsorbed on aluminum phosphate has an adsorption percentage of at least 70%.

According to the tenth embodiment of the present invention, the Hib antigen used in the combination vaccine of the present invention is derived from the capsular polysaccharide of Haemophilus influenzae type b (Hib) strain 760705.

According to one aspect of the tenth embodiment, the Hib PRP antigen is combined with a carrier protein selected from the group consisting of but not limited to the following carrier proteins: CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, tetanus Toxoid fragment C, pertussis toxoid, Haemophilus influenzae protein D, Escherichia coli LT, Escherichia coli ST and Pseudomonas aeruginosa exotoxin A, outer membrane complex c (OMPC), porin, transfer iron Protective properties of protein binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesive A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, and Bacillus anthracis Antigen (PA) and detoxification edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and tuberculosis Purified protein derivatives (PPD), synthetic peptides, heat shock proteins, pertussis protein, cytokine, lymphoid factor, hormones, growth factor, and a variety of human CD4+T cell epitopes containing antigens derived from multiple pathogens such as N19 Artificial protein, iron uptake protein, toxin A or B from Clostridium difficile and Streptococcus agalactiae protein.

Also preferably, Hib PRP and tetanus toxoid (TT) are combined by the following: CNBr chemistry, reductive amination chemistry, cyanation chemistry or has been published in New York: Marcel Dekker, 1994: 37-69, Kniskern et al. "Conjugation: design, chemistry, and analysis", and any other chemistry disclosed in "Development and clinical uses of Haemophilus influenzae type B conjugate vaccines" by Ellis et al.

According to the second aspect of the tenth embodiment, the carrier protein is present in the composition of the present invention in a free form and an conjugated form. The unconjugated form preferably does not exceed 20% of the total amount of the carrier protein in the composition as a whole. It is more preferably less than 5% by weight.

According to the third aspect of the tenth embodiment, the Hib antigen is basically not adsorbed on any adjuvant.

According to the fourth aspect of the tenth embodiment, the Hib antigen can be adsorbed on any adjuvant unintentionally or intentionally.

According to the fifth aspect of the tenth embodiment, the adsorption percentage of Hib antigen on any adjuvant is less than 20%.

According to the eleventh embodiment of the present invention, the whole cell pertussis (wP) antigen preparation used in the combined vaccine composition of the present invention is preferably mixed with Bordetella pertussis strains 134, 509, 25525 and 6229 in a specific ratio And then it was made by inactivating by using an improved inactivation method without thimerosal, resulting in decreased reactogenicity and increased efficacy, and the wP antigen may or may not be adsorbed on the aluminum-based adjuvant.

According to one aspect of the eleventh embodiment, the whole-cell pertussis (wP) antigen preparation used in the combination vaccine composition of the present invention is preferably composed of Bordetella pertussis strains 134, 509, 25525 and 6229. :1:0.25:0.25 ratio is mixed and made.

According to the second aspect of the eleventh embodiment, one or more of the following inactivation treatments are used to inactivate the whole cell pertussis (wP) antigen preparation used in the combination vaccine composition, and the inactivation treatment includes heating, UV , Formalin/Formaldehyde, Acetylethyleneimine, etc.

Still preferably, a combination of heat treatment and chemical treatment is used to inactivate the whole cell pertussis (wP) antigen preparation used in the combination vaccine composition. Still preferably, heating and inactivating in the presence of formaldehyde at 56±2°C for 10 to 15 minutes, wherein the wP blocks remain non-agglomerated and are easy to homogenize, resulting in reduced reactogenicity and providing better Good wP effectiveness.

According to the third aspect of the eleventh embodiment, the whole-cell pertussis (wP) antigen preparation used in the combined vaccine composition may or may not be adsorbed on an aluminum-based adjuvant, such as aluminum hydroxide, aluminum phosphate or a combination thereof (eg Before or after mixing with other components (if present)). If adsorbed, one or more wP strains (ie 134, 509, 25525, and 6229) can be adsorbed individually or together as a mixture.

According to the twelfth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the thirteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the fourteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the fifteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the sixteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the seventeenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the eighteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the nineteenth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twentieth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-first embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-second embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-third embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-fourth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-fifth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-sixth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-seventh embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-eighth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the twenty-ninth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the thirtieth embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the thirty-first embodiment of the present invention, the multi-dose combination vaccine composition/preparation includes:

According to the thirty-second embodiment of the present invention, the final single-dose combination vaccine composition/preparation includes:

According to the thirty-third embodiment of the present invention, the final single-dose combination vaccine composition/preparation includes:

According to the thirty-fourth embodiment of the present invention, the final single-dose combination vaccine composition/preparation includes:

According to the thirty-fifth embodiment of the present invention, the final single-dose combination vaccine composition/preparation includes:

NMT-no more than

According to the thirty-sixth embodiment of the present invention, one or more antigens of the final combined vaccine composition can be substantially not adsorbed on any adjuvant.

According to the thirty-seventh embodiment of the present invention, the pH of the immunogenic composition may be in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 6.0 to pH 7.5; still more preferably in the range of pH 6.2 to Within the range of pH 7.2; optimally within the range of pH 6.3 to pH 6.8.

According to the thirty-eighth embodiment of the present invention, the immunogenic composition may additionally comprise selected from carbonate, phosphate, acetate, succinate, borate, citrate, lactate, gluconate and tartrate The composition group of buffers, and more complex organic buffers including phosphate buffers, the phosphate buffers include sodium phosphate and/or potassium phosphate, the ratio of which is selected to achieve the desired pH. In another example, the buffer contains Tris (hydroxymethyl)aminomethane or "Tris" formulated to achieve the desired pH. In yet another example, the buffer can be a minimum essential medium containing Hanks' salt. The present invention also contemplates other buffers such as HEPES, Pipazine-N, N'-bis (PIPES) and 2-ethanesulfonic acid (MES). The buffer helps stabilize the immunogenic composition of the invention. The amount of the buffer can be in the range of 0.1 mM to 100 mM, preferably selected from 5 mM, 6 mM, 7 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM.

In yet another aspect of the embodiment, the immunogenic composition may additionally include a pharmaceutically acceptable excipient selected from the group consisting of surfactants, polymers, and salts. Examples of surfactants may include nonionic surfactants such as polysorbate 20, polysorbate 80, and the like. Examples of polymers may include polydextrose, carboxymethyl cellulose, hyaluronic acid, cyclodextrin and the like. Examples of the salt may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, MgCl2, and the like. Preferably, the salt may be NaCl. Generally, the amount of salt can be in the range of 100 mM to 200 mM.

Amino acids such as histidine, glycine, arginine, and lysine can be added to stabilize the immunogenic composition.

According to the thirty-ninth embodiment of the present invention, the immunogenic composition may additionally include one or more selected from aluminum salts (Al ³⁺ ), such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ) , Alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvants, liposomes or adjuvants in the group of oil-in-water emulsions.

Still preferably, the composition contains aluminum phosphate (AlPO ₄ ) as an adjuvant.

Still preferably, the composition contains aluminum hydroxide (AlOH ₃ ) as an adjuvant.

In one aspect of the thirty-ninth embodiment, the antigen of the final preparation can be adsorbed on the in-situ aluminum phosphate gel or the ready-made aluminum phosphate gel or a combination thereof.

In a preferred aspect of the thirty-ninth embodiment, the composition of the present invention may contain an amount of 2.5 mg/0.5 ml or less, particularly an amount of 1.5 mg/0.5 ml to 0.1 mg/0.5 ml. Agent.

According to the fortieth embodiment of the present invention, the immunogenic composition may additionally include an immunostimulatory component selected from the group consisting of oil and water emulsion, MF-59, liposome, lipopolysaccharide, saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacyl monophosphoryl lipid A, AS01, AS03, oligonucleotides, oligonucleosides containing at least one unmethylated CpG and/or liposome Acid, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p 1005, CRL-8300 Adjuvant, mural dipeptide, TLR-4 agonist, flagellin, flagellin derived from gram-negative bacteria, TLR-5 agonist, fragment of flagellin capable of binding to TLR-5 receptor, α- C-galactosidyl ceramide, chitosan, interleukin-2, QS-21, ISCOMS, squalene mixture (SAF-1), Quil A, cholera toxin B subunit, polyphosphazene and derivatives , Mycobacterial cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, combination of saponin and sterols and lipids.

According to the forty-first embodiment of the present invention, the immunogenic composition may additionally comprise selected from the group consisting of Phemerol chloride, phenol, m-cresol, thimerosal, formaldehyde, aniline chloride, benzyl alcohol, chloroprene A preservative of the group consisting of alcohol, p-chloro-m-cresol, or benzyl alcohol or a combination thereof. The vaccine composition may contain a preservative for a single immunization, or may contain a preservative for multiple immunizations (ie, a "multi-dose" kit). The preservative is preferably contained in multiple doses. As an alternative (or in addition) to including a preservative in a multi-dose composition, the composition can be contained in a container with a sterile adapter for removing material. Generally, the amount of preservative can be in the range of 0.1 mg to 50 mg.

According to the forty-second embodiment of the present invention, depending on the route of administration and the desired formulation, the immunogenic composition may additionally contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling agents or thickening agents , Flavors, colors, etc.

According to the forty-third embodiment of the present invention, the immunogenic composition may be all liquid, but is not limited thereto. Suitable forms of liquid preparations may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions, and elixirs buffered to the desired pH.

The immunogenic composition of the present invention may be in the form of a transdermal preparation, including lotion, gel, spray, ointment or other suitable techniques. If nasal or respiratory (mucosal) administration is required (for example, aerosol inhalation or insufflation), the composition may be in a certain form and be dispensed by a squeeze spray dispenser, pump dispenser, or aerosol dispenser. Aerosols usually rely on hydrocarbons under pressure. The pump dispenser can preferably dispense metered doses or doses with specific particle sizes. When in the form of solutions, suspensions and gels, in some embodiments, the immunogenic composition contains a large amount of water (preferably purified water) in addition to the active ingredient.

According to the forty-fourth embodiment of the present invention, the combination vaccine can be stable at 2-8°C for 12 to 36 months; at 25°C for 2 to 6 months; at 37°C for 1 week to 4 weeks.

According to the forty-fifth embodiment of the present invention, the immunogenic composition can be formulated as a method for reducing or preventing the onset of health conditions including diphtheria, tetanus, pertussis, hepatitis B virus, and influenza B Haemophilus, poliovirus infection, the method involves parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injection or sustained release from implants or by eye drops Administration or nasal cavity or rectum or buccal or vagina, oral or stomach or mucosa or tongue, alveolar or gum or olfactory or respiratory mucosal administration or any other immunization route, administering an immunologically effective amount of immunity to human subjects Original composition.

According to the forty-sixth embodiment of the present invention, the immunogenic composition can be formulated into a single-dose vial or a multi-dose vial (2-dose or 5-dose or 10-dose vial) or a multi-dose kit or pre-filled syringe, wherein The immunogenic composition can be administered in a single-dose schedule, or preferably a multiple-dose schedule, wherein after the main vaccination method, if necessary, 1-3 separate doses are given at subsequent intervals after 1-3 years . The dosage regimen will also be determined, at least in part, based on the need for the booster dose required to confer protective immunity.

Still preferably, according to a two-dose regimen that includes a first dose and a second dose administered at a subsequent time interval after 1-3 years, the immunogenic composition can be formulated to be administered to people 2 years of age or younger. Or children.

Still preferably, the immunogenic composition can be administered simultaneously with other drugs or any other vaccines.

According to the forty-seventh embodiment of the present invention, the applicant found that considering: i) the method of preparing a single antigen, ii) the order of adding the antigen, iii) using a specific amount of a specific adjuvant for certain antigens, iv) an antigen Single or combined adsorption on the adjuvant, v) the degree of adsorption of the antigen on the adjuvant, vi) use the lowest alum concentration, vii) use the optimal concentration and type of preservative, and viii) use various parameters, including stirring , Temperature and pH, when the vaccine is produced by the method disclosed below, a multi-dose whole liquid combination vaccine with improved immunogenicity and reduced reactogenicity can be obtained. The biological source of the strain used in the SIIPL combination vaccine: Diphtheria toxoid:

The Corynebacterium diphtheriae PW8 CN2000 strain was obtained in 1973 in a freeze-dried form from the Central Research Institute (CRI) of the Kasauli State Control Bureau, Himachal Pradesh, India, from the Wellcome Research Laboratory in London, England. The strain was revived under the main seed batch-Corynebacterium diphtheriae CN2000 A1 at CRI Kasauli and further lyophilized. Tetanus toxoid:

The Clostridium tetani Harvard strain 49205 strain was obtained in freeze-dried form by the National Control Agency CRI Kasauli from the Riksvolks Gzonheide Institute (Netherlands). pertussis:

Four Bordetella pertussis strains 134, 509, 6229 and 25525 are required to manufacture pertussis vaccine in SIPL. The main seeds of strains 134 and 509 came from the Ricks Institute in the Netherlands and were obtained from the Central Institute of the Kasauli State Control Bureau, Himachal Pradesh, India. The main seeds of strains 6229 and 25525 were from the Liszt Institute in England. Hepatitis B:

Rhein Biotechnology (Germany) constructed a recombinant Hansenula strain containing HBsAg surface antigen gene. Rheinland Biotech also produced a master cell bank (MCB Hansenula K3/8-1 strain ADW, 12/94), and performed all characterization tests on the bank. Haemophilus influenzae type b:

The source organism that produces the cell substrate is Haemophilus influenzae type b, strain 760705. This strain was first isolated from a baby boy (born 14-8-74) who was 2 years and 2 months old in November 1976. The strains were passaged three times at the Academic Medical Center of the University of Amsterdam (AMC) and then stored at -70°C. As part of the cooperation between SIIPL and the Netherlands Vaccine Institute (NVI), this strain has been transferred to SIIPL. IPV :

The strain and source of Salk polio virus are as follows. Poliomyelitis virus type 1 : Strain: Mahoney Source: Birsofen Biotech, Netherlands Poliovirus type 2 : Strain: MEF1 Source: Birsofen Biotech, Netherlands Poliovirus Type 3 : Strain: Saukett Source: Birr, Netherlands Sofin Bio

Throughout the specification, the word "comprise" or variants such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or elements, integers or Step groups, but do not exclude any other elements, integers or steps or elements, integers or step groups.

The use of the expression "at least" or "at least one" indicates that one or more elements or ingredients or quantities are used because they can be used to achieve one or more desired purposes or results in the embodiments of the present invention. Although certain embodiments of the present invention have been described, these embodiments are given by way of example and are not intended to limit the scope of the present invention. After reading the disclosure herein, those skilled in the art can change or modify the formulation of the present invention within the scope of the present invention. Such changes or modifications are completely within the spirit of the present invention.

The numerical values given for various physical parameters, sizes and quantities are only approximate values, and it is conceivable that, unless otherwise stated in the specification, values higher than the numerical values assigned to physical parameters, dimensions and quantities fall within the scope of the present invention Inside.

Although the text has focused on the specific features of the preferred embodiment, it should be understood that many additional features can be added and many changes can be made in the preferred embodiment without departing from the principles of the invention. From the disclosure herein, these and other changes in the preferred embodiment of the present invention will be obvious to those skilled in the art, and it will be clearly understood that the foregoing descriptive content should only be interpreted as It is an explanation rather than a limitation to the present invention. advantage

The invention described above has several technological advances and advantages, including but not limited to the realization of a combination vaccine composition comprising D, T, wP, HBsAg, Hib PRP-TT conjugate and IPV and its manufacturing method. When compared with other combination vaccine compositions, the present invention provides the following advantages: 1. Full-liquid combination vaccine 2. Compared with the standard dose, the dose of IPV antigen is reduced, which shows that it is compared with the standard dose (40-8-32 DU) Equivalent efficacy 3. Improved immunogenicity of D, T, wP, HepB, Hib, IPV antigens 4. Improved stability tested at 2-8°C and room temperature within 12 months 5. High-purity diphtheria toxoid (D) and tetanus toxoid (T) produced using semi-synthetic media that does not contain infectious spongiform encephalopathy (TSE) or bovine spongiform encephalopathy (BSE). 6. Whole-cell Bordetella pertussis (wP) antigen contains 1:1:0.25:0.25 ratio of Bordetella pertussis strains 134, 509, 25525 and 6229, thereby improving the efficacy and immunity against Bordetella pertussis Originality. 7. An improved method to inactivate the components of the whole cell of Bordetella pertussis (wP) using a combination of heat and formaldehyde inactivation. This method does not contain thimerosal and the inactivated whole-cell pertussis antigen remains non-clumping and homogeneous, resulting in reduced reactogenicity and providing better efficacy over a longer period of time. 8. The free PRP in the total amount of Haemophilus influenzae type b PRP-TT conjugate is low (less than 7%). 9. The adsorption percentage of Hib antigen on any adjuvant is less than 20%. 10. The adsorption characteristics of the diphtheria toxoid antigen (D), tetanus toxoid (T) antigen and hepatitis B (HepB) surface antigen adsorbed separately on aluminum phosphate adjuvant have been improved, thereby improving the efficacy and immunogenicity. 11. The minimum total alum content (Al ³⁺ ) to ensure reduced reactivity. 12. The optimized concentration of 2-phenoxyethanol (2-PE) and at least one paraben (methyl paraben or propyl paraben) are used as preservatives, thus effectively maintaining the The antibacterial ability of a full-dose combination vaccine. Instance

The following examples are included to illustrate the preferred embodiments of the present invention. Those familiar with the art should understand that the compositions and techniques disclosed in the following examples represent the techniques discovered by the inventor to play a good role in the implementation of the present invention, and therefore can be considered as a better way to implement them. However, according to the present invention, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed, and similar or similar results can still be obtained without departing from the spirit and scope of the present invention. Example 1 : Various combinations of vaccine compositions according to the present invention

In addition, the pH of the composition as described above was adjusted to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate, and the volume was made up by adding physiological saline (0.9%). The vaccine may contain traces of glutaraldehyde, formaldehyde, neomycin, streptomycin, and polymyxin B used in the manufacturing process.

In addition, the pH of the composition as described above was adjusted to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate, and the volume was made up by adding physiological saline (0.9%). The vaccine may contain traces of glutaraldehyde, formaldehyde, neomycin, streptomycin, and polymyxin B used in the manufacturing process. Example 2 : Manufacturing method of Haemophilus influenzae type b conjugate

The flowchart in Figure 1 shows an overall view of the manufacturing steps. Each of the 53 steps of the method is briefly introduced as follows: Step 1 : Inoculation stage I shake flask (S1) :

Use the working seed batch vial to inoculate the inoculation stage shake flask containing 0.22 µm filtered seed medium. Use a disposable PETG 125 mL flask with a working volume of 25 mL. This stage is carried out under controlled stirring (200±50 rpm) and temperature (36±2°C) in a constant temperature oscillator. After reaching proper bacterial growth (OD590≥1.0), the culture is transferred to the next inoculation stage (S2 stage), which is as described in step 2. Gram staining was performed as a control in the method to ensure the purity of the culture (Gram-negative cocci). Step 2 : Inoculation phase II shake flask (S2) :

The S2 inoculation stage consists of 2 L fernbach flasks (S2A and S2B) with a working volume of 800 mL. The S2A flask was used for OD590 measurement until the OD590 was within acceptable standards, and the S2B flask was used for the inoculation of the S3 stage. Both flasks are filled with filter-sterilized medium, which is the same as the S1 inoculation stage. The S1 stage flask was used for the inoculation stage II shake flask. This stage is carried out in a constant temperature oscillator under controlled stirring (200±50rpm) and temperature (36±2°C). After reaching proper bacterial growth (OD ₅₉₀ ≥1.0), the culture is transferred to the next inoculation stage (S3 stage), which is as described in step 3. Gram staining was performed as a control in the method to ensure the purity of the culture (Gram-negative cocci). Step 3 : Inoculation stage III fermentor:

The S3 inoculation stage includes a 120 L fermentation tank with a 35 L working volume. The fermentation tank is filled with the same medium as the previous inoculation stage. The S2 stage flask was used to inoculate the fermentor. In the inoculated fermenter, the growth is carried out under temperature (36±2℃), DO (10% set value), stirring (300-600 rpm), aeration (1-5 LPM) and back pressure (0.2 bar). After reaching proper bacterial growth (OD590≥1.0), the culture is transferred to the next production stage (stage S4), which is as described in step 4. Gram staining was performed as a control in the method to ensure the purity of the culture (Gram-negative cocci). Step 4 : 1200 L scale production fermentation:

The working volume of 1200 L production fermenter is 800 L. It is filled with basal medium components and steam sterilized in situ. Subsequently, various medium supplements were added after passing through a 0.22 μm filter. Inoculate the fermentor with the S3 stage culture obtained from step 3. Fermentation is controlled under controlled dissolved oxygen (20%-set point), temperature (36±2℃), pH (7.1-7.4), stirring (40-400 rpm), aeration (50-300 LPM) and back pressure ( 0.2 bar). Two discrete nutrients are added in the fermentation process. The growth is monitored by measuring OD590 (OD590≥3.5), and the fermentation is considered complete after reaching the stable stage. During the growth and resting phases, polysaccharide products are secreted and accumulated in the culture medium. Gram staining was performed as a control in the method to ensure the purity of the culture (Gram-negative cocci). Step 5 : Formalin treatment:

By using chemical reagents (formalin), the bioburden can be reduced in this step. Add 0.1% formalin, and incubate the fermentation broth at 37°C for not less than 2 hours. After the formalin treatment, the container was quickly cooled to <15°C. It has been verified that adding formalin can reduce the bioburden. After the incubation period, use the culture plate for verification. The broth with reduced bioburden can be prepared for harvest as described in step 6. Step 6 : Continuous centrifugal harvest:

Continuous centrifugation is used as the main harvesting step. This step is performed to separate the crude broth containing polysaccharides from the inactivated biomass. The purpose of using a continuous centrifuge is to remove >90% of the biomass, as measured by OD590 reduction. The centrifuge operates with a liquid flow rate of approximately 15000 g and 200-500 L/h. The centrifuged supernatant was further processed as described in step 7. Step 7 : 50LP deep filtering:

The centrifuged supernatant is passed through a 50LP depth filter to remove coarse materials such as cell debris. This step allows the product to pass through the filtrate and is connected in series with other depth filters, as described in step 8. Step 8 : 90LP deep filtering:

The filtrate from the 50LP depth filter further passes through the 90LP depth filter (nominal 0.22 µm grade) to further remove any insolubles that may not have been retained by the previous depth filter. This step ensures that the filtrate is basically free of cell debris and can pass through the 0.22 µm filter smoothly. The subsequent filtering steps are described in step 9. Steps 9 and 10 : 0.22 µm filtration:

The filtrate from the 90 LP depth filter is further passed through a 0.22 µm filter, and the filtrate is collected in a storage tank. Steps 11 and 12 : 100 kD concentration and diafiltration:

This step is performed to remove medium components and small molecular weight impurities. In addition, concentration is performed to reduce the workload. Because the molecular weight of Hib polysaccharide (PRP) is ≥500 kD, a 100 kD molecular weight cut-off was selected. The broth is concentrated to about 10 times, and then diafiltration is not less than 5 volumes using 0.01 M PBS buffer (pH 7.2). The product obtained from the retentate is called "crude PRP" and is further processed as described in step 13. The concentrated broth is transferred to the DSP area through the transfer port through a 0.22 µm filter to ensure that no bacteria are transferred to the DSP area. Step 13 : CTAB precipitation:

CTAB (hexadecyl trimethyl ammonium bromide) is a cationic detergent used to precipitate polysaccharides. CTAB is composed of hydrophilic regions and hydrophobic parts, and precipitates proteins, nucleic acids and polysaccharides. The crude PRP obtained from step 12 was precipitated at a concentration of 1% CTAB and incubated for> 2 hours. In step 14, CTAB precipitation harvesting is described. Steps 14 , 15 and 16 : CTAB precipitation centrifugation, collection and storage:

In SEZ-3, FF, the CTAB pellet was centrifuged at 15000 rpm using a continuous centrifuge. The CTAB pellet was harvested, weighed, aliquoted and stored at ≤-20°C for further processing. This is the holding step in the first method. Steps 17 and 18 : Thaw and dissolve the CTAB paste:

Thaw the frozen CTAB paste to room temperature. Dissolve the thawed precipitate in 5.85% NaCl solution. The dissolution is carried out in a stirred tank, and the polysaccharide product is dissolved in the water phase. The tank contains some insoluble substances, which are derived from precipitated proteins and nucleic acids. The suspension is further processed as described in step 19. Step 19 : Centrifuge:

Centrifuge the material obtained in step 18 at 2-8°C and 5000-6500 rpm for 20-30 minutes to remove undissolved material. Collect the centrifugal supernatant and proceed as described in step 20 for further processing. Step 20 : 72% ethanol precipitation:

72% ethanol was used to precipitate PRP. Using 96% ethanol produces a final concentration of 72% ethanol relative to the supernatant obtained in step 19. The precipitation takes place overnight at 2-8°C. Harvest the resulting pellet as described in step 21. Steps 21 and 22 : Centrifugation and precipitation dissolution:

The 72% ethanol precipitate was collected by centrifugation at 2-8°C and 5000-6500 rpm for 20-30 minutes. Dissolve the resulting precipitate in WFI until a clear visual effect is obtained. In step 23, the subsequent treatment of dissolving the precipitate is described. Step 23 : DOC and 32% ethanol precipitation:

To the material obtained from step 22, add 6% sodium acetate and 1% sodium deoxycholate (DOC). Use 96% ethanol to produce a final concentration of 32% ethanol. Both DOC and 32% ethanol promote the precipitation of protein impurities while keeping polysaccharides in the liquid phase. The precipitation is carried out at 2-8°C overnight (not less than 8 hours). Step 24 : Centrifuge:

Centrifuge the material obtained in step 23 at 2-8°C and 5000-6500 rpm for 20-30 minutes to remove the precipitate. Collect the centrifuge supernatant and proceed as described in step 25 for further processing. Step 25 : Depth and carbon filtration:

In step 24, the supernatant obtained contains soluble PRP, and is subjected to deep filtration and then carbon filtration to remove nucleic acids and pigments. The removal of nucleic acids was monitored by intermittently measuring absorbance (A ₂₆₀ ) at 260 nm. After reaching target A ₂₆₀ , the solution is filtered through a 0.22 µm filter, and then the filtered solution is further processed as described in step 26. Step 26 : 64% ethanol precipitation:

The filtered material obtained in step 25 is further precipitated with 96% ethanol, and the final concentration is 64% ethanol. The precipitation takes place overnight at 2-8°C. The precipitate obtained is collected by centrifugation and further processed as described in step 27. Step 27 : Precipitation collection and dissolution:

Pour out the supernatant and discard to collect the precipitate. The precipitate was dissolved in WFI at room temperature. Step 28 : 300 kD concentration and diafiltration:

Use 300 kD NMWCO membrane to concentrate the dissolved precipitation solution. Use WFI to further percolate it no less than (NLT) 8 times. Follow step 29 to further process the resulting retentate. Steps 29 and 30 : 0.22 µm filtered and purified PRP storage:

The 300 kD UF retentate was passed through a 0.22μm filter as a clarification step to minimize the bioburden. The purified PRP obtained is divided into aliquots and stored at ≤-20°C until further use as described in step 31. Send the purified PRP sample to QC for analysis. Step 31 : Thaw and merge:

Based on the batch size of the conjugate, the appropriate amount of natural polysaccharide obtained from step 30 is thawed. The PRP content of the combined substance is tested, which is necessary for further processing as described in step 32. Step 32 : 100 kD concentration:

The combined purified polysaccharide must have the lowest concentration (8-12 mg/mL) for further processing. If the combined polysaccharide concentration is lower than the target concentration, use a 100 kD UF NMWCO membrane to concentrate the combined polysaccharide solution.

After concentration, a sample is drawn to ensure that the lowest concentration is reached for the subsequent steps (step 33). Step 33 : Alkaline depolymerization:

Under mild alkaline conditions using carbonate-bicarbonate buffer, the concentrated polysaccharide (equivalent to 74g/110g) obtained from step 32 was depolymerized. After reaching the target polysaccharide size, activate the depolymerized polysaccharide as described in step 34.

Step 34: Start of polysaccharide:

The depolymerized polysaccharide obtained in step 33 is activated with cyanogen bromide. This activation is performed under a nitrogen atmosphere. Cyanogen bromide is a highly toxic chemical, so be careful when handling this chemical. Step 35 : Connector connection:

Within 6-10 minutes, the freshly prepared dihydrazine adipate (ADH) solution is added to the reaction mixture obtained from step 34. The reaction is carried out at 2-10°C for not less than 16 hours. The function of the ADH linker is to provide the amine group required for the conjugation reaction in the polysaccharide. Step 36 : Concentration and diafiltration:

The reaction mixture obtained from step 35 was concentrated and diafiltered volume by volume with phosphate buffered saline (PBS) using a 10 kD NMWCO UF membrane to remove free ADH. The removal of ADH was monitored on HPLC, and diafiltration was continued until the free ADH level reached below 5%. The obtained retentate was further diafiltered with not less than 5X MES-NaCl buffer. It is further concentrated to reach a concentration of not less than 20 mg/mL. The concentrated processed PRP is kept at 2-8°C until further use as described in step 37. Steps 37 and 38 : 0.22 µm filtered and processed PRP storage:

The retentate from step 36 is passed through a 0.22 µm filter, which is used as a clarification step. This also ensures that the bioburden level is controlled in the method, which is performed in the C-level area. Collect filtered activated polysaccharides, sample, aliquot and store at 2-8°C until further processing. Samples were extracted from the processed polysaccharide pool for analysis, which included PRP molecular size (kD), PRP content and PRP activation degree. In step 40, further processing of the processed PRP is described. Step 39 : TT 10 kD concentration and diafiltration:

The conjugation reaction requires two components, the processed polysaccharide and the carrier protein (TT). The carrier protein was concentrated and diafiltered with MES-NaCl buffer using a 10 kD UF NMWCO membrane. Then use the same membrane to further concentrate the diafiltered carrier protein to not less than 20 mg/mL. Step 40 : Join:

The conjugation reaction requires two components, the processed polysaccharide and the carrier protein (TT). From step 38, an activated polysaccharide component is obtained. The carrier protein is obtained from step 39. In the presence of EDC, the two components are mixed in an appropriate amount at a ratio of PRP:TT=1:1 (w/w) under stirring. The conjugation reaction was monitored on HPLC and continued until ≥85% protein conversion rate (based on the conversion rate of free protein to conjugate) was reached. Step 41 : Quenching:

After the joining reaction has progressed to the acceptable standard of its conversion (step 40), the reaction is terminated by quenching. The joining reaction was quenched with phosphate EDTA buffer. This bonding reaction is then processed as described in step 42. Step 42 : 30 SP and 0.22 micron filter:

The conjugate obtained from step 41 is filtered through a 30 SP filter, and then filtered at 0.22 μm. This ensures that any large aggregates are removed. Treat the filtered conjugate as described in step 43. Step 43 : 300 kD ultrafiltration and percolation:

Using a 300 kD UF NMWCO membrane, the ligation reaction mixture obtained in step 42 was diafiltered with 0.05% saline. Diafiltration was performed to remove the binding reagent and unreacted TT. The resulting retentate is further processed as described in step 44. Steps 44 and 45 : 0.22 µm filtration and storage of crude conjugate:

The retentate from step 43 was passed through a 0.22 µm filter, which was used as a clarification step. This also ensures that the bioburden level is controlled in the method, which is performed in the C-level area. The filtered crude conjugate was collected, sampled and stored at 2-8°C until further processing. In step 46, the further processing of the crude joint is described. Step 46 : Dilution of crude conjugate:

If necessary, the crude conjugate from step 45 is diluted with WFI to reach the target concentration of 4±1 mg/mL, and is further processed by the precipitation step described in step 47. Step 47 : Precipitation of ammonium sulfate:

The diluted conjugation reaction mixture was further processed using ammonium sulfate (50% w/v stock solution) to remove free PRP. Under stirring, the precipitation step is performed at less than 15°C. The precipitation step brings the conjugate into the precipitate and leaves free PRP in the supernatant. After the addition of ammonium sulfate, the resulting suspension was stored at less than 15°C for not less than 12 hours without stirring. Step 48 : precipitation collection and dissolution:

Centrifuge the suspension from step 47 at about 7000 g for 40±10 minutes at 2-8°C. The supernatant was discarded by decantation, and the obtained precipitate was dissolved in Tris-saline. Step 49 : 300 kD percolation:

The resulting solution from step 48 was filtered through a 30 SP depth filter and diafiltered with 20 mM Tris-saline using a 300 kD NMWCO membrane. Step 50 : GPC chromatographic purification:

The resulting solution from step 49 was loaded onto an approximately 70 L GPC column for size exclusion chromatography, which contained a Toyopearl HW-65F hydroxylated methacrylic polymer bead gel. The use of GPC chromatography (after ammonium sulfate) on the treated conjugate can reduce the free PRP content in the resulting material. The column was eluted with 20 mM Tris 0.9% NaCl, and fractions were collected based on A280. The appropriate fractions are combined based on acceptance criteria for free PRP, ratio and molecular size, and the combined is further processed as described in step 51. Step 51 : 300 kD percolation:

Using a 300 kD UF NMWCO membrane, the combined conjugate eluate from step 50 was diafiltered with 20 mM Tris. The target for the volume of the retentate is to have a PRP content of about 1 mg/mL. Steps 52 and 53 : 0.22 µm filtration:

In a Class A environment, filter the whole conjugate obtained in step 51 through a 0.22 μm filter to ensure sterility. The 0.22 µm filter is tested for integrity. The sample from the filtered whole conjugate is sent to QC for complete analysis. The filtered conjugate is labeled as "sterile Hib integral conjugate" and stored at 2-8°C. The whole joint will be stored at 2-8°C for up to 3 months, after that, if it is not used, it can be stored at -70°C for up to 1 year. The quality characteristics of the obtained Hib PRP-TT conjugate antigen are as follows: PRP content (µg/0.5ml): 8.1 Ratio (PRP:TT): 0.5 Free PRP(%): 4.8% PMW(kD): 983 Average MW(kD) ): 752 Example 3 : Method of manufacturing inactivated wP antigen Method of inactivating whole cell pertussis (wP) antigen:

After performing various experiments, the inactivation method was optimized. These experiments include inactivation at 56°C for 10 min in the presence of formaldehyde, inactivation at 56°C for 15 min in the presence of formaldehyde, and inactivation at 56°C for 10 min in the presence of hymine min, inactivation in the presence of hymine at 56°C for 15 min and heating at 56°C for only 30 min. No significant difference in efficacy was observed with these methods. In these methods, the choice of 56°C for 10 minutes in the presence of formaldehyde is because the pertussis cell mass produced by this method is more homogeneous than the other methods mentioned above. The manufacturing method of inactivated wP antigen includes the following steps: a). Bordetella pertussis strain 134 is inactivated at 56°C for 10-15 minutes in the presence of formaldehyde b). Bordetella pertussis strain 509 at 56°C in the presence of formaldehyde Inactivate at ℃ for 10-15 minutes c). Bordetella pertussis strain 25525 and 6229 are inactivated at 56℃ for 10-15 minutes in the presence of formaldehyde c). Bordetella pertussis strain 6229 is killed at 56℃ in the presence of formaldehyde Live for 10-15 minutes d). Then mix and inactivate the Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25. e). Adsorbed on aluminum-based adjuvant as appropriate.

This method does not contain thimerosal and the inactivated whole-cell pertussis antigen remains non-clumping and homogeneous, resulting in reduced reactogenicity and providing better efficacy over a longer period of time. Example 4 : Production method of inactivated poliovirus (IPV) 1. Poliomyelitis virus can be grown by the following methods: a) CCL81-VERO (monkey kidney) cells are used for poliovirus, namely Sabin and Salk The host cell where the strain grows. b) After infecting the host cells with the desired poliovirus strain and incubating for 72 hours, the media containing the virus and cell debris are combined and collected in a single container. c) The filtrate was subjected to tangential flow filtration with a 100 KDa filter cartridge; diafiltered with phosphate buffer, and purified by anion exchange chromatography. d) Before administering to patients, the virus must be inactivated using an appropriate inactivation method. 2. Formalin inactivation includes the following steps: a) The purified virus pool is buffer-exchanged from phosphate buffer to Tris buffer in the pH range of 7 to 7.5 (30 to 50 mM), b) adding to the above mixture M-199 medium containing glycine (5 gm/l) c) add 0.025% formaldehyde, and then mix, d) then incubate the mixture at 37°C for 5 to 13 days while continuously stirring the virus mass on a magnetic stirrer, e ) On the 7th day, place the incubated mixture in an intermediate TFF system (100 KDa, 0.1 m2), and perform final filtration after inactivation. f) Then store the filtered material at 2-8°C, g) perform D -Ag ELISA is used to determine the D-Ag unit h) Unit price pool materials of IPV type 1, type 2 and type 3 are then mixed to form trivalent or divalent IPV (Salk or Sabin serotype) i) Adjust the pH of the final preparation, and A final formulation with a pH between 6 and 6.8 is obtained. j) IPV antigen (Sabin or Salk strain) is then added to the final combined vaccine composition and adsorbed on the adjuvant (aluminum phosphate) present in the combined vaccine. It is found that the adsorption percentage of IPV antigen to IPV type 1 is 10 In the range of %-30%, IPV 2 type is in the range of 60%-100%, and IPV 3 type is in the range of 0%-25%. 3. The preparation procedure when IPV (Sabin and Salk strains ) are respectively adsorbed on aluminum salt: a) Take the required volume of autoclaved AlPO4 to make the final alum (Al ³⁺ ) concentration between 0.1 and 0.8 in a 50 ml container mg/dose between b) add IPV material with adjusted D-Ag unit, and make up the volume with diluent (10x M-199 + 0.5% glycine), c) adjust the pH of the final formulation, and obtain a pH between 6 and The final formulation between 6.8. 4) The unit price pool with aluminum adsorption is correspondingly formulated into trivalent or divalent IPV (Salk or Sabin serotype ). Results: The adsorption percentage of IPV 1, 2 and 3 (Sabin&Salk) on aluminum phosphate (AlPO4) salt is found At least 90% of applicants have been able to reduce the dose of poliovirus antigen by 2 times (and the standard dose of poliovirus antigen is type 1-40 DU, type 2-8DU, type 3-32DU).

實例 5 ：製造聯合疫苗之方法 該實例簡要介紹包含 D 、 T 、 wP 、 HBsAg 、 Hib PRP-TT 接合物、 IPV 及防腐劑之聯合疫苗組合物之製造方法： 組分 I- 經明礬吸附之白喉類毒素 組分 II- 經明礬吸附之破傷風類毒素 組分 III–wP 抗原 ( 如實例 3 中所揭示 ) 組分 IV- 經明礬吸附之乙型肝炎表面抗原 組分 V-Hib PRP 接合物 ( 如實例 2 中所揭示 ) 組分 VI-IPV 抗原 ( 如實例 4 中所揭示 ) 1. 製備包含經明礬吸附之白喉類毒素之組分 I ： a). 將磷酸鋁轉移到容器/器皿中 b). 添加白喉類毒素 c). 用醋酸/氫氧化鈉將pH調節至4.5至5.5 d). 等待穩定 e). 用氫氧化鈉/碳酸鈉將pH調節至5.5至6.5 f). 等待穩定2. 製備包含經明礬吸附之破傷風類毒素之組分 II ： a). 將磷酸鋁轉移到容器/器皿中 b). 添加破傷風類毒素 c). 用醋酸/氫氧化鈉將pH調節至4.5至5.5 d). 等待穩定 e). 用氫氧化鈉/碳酸鈉將pH調節至5.5至6.5 f). 等待穩定3. 製備包含經明礬吸附之乙型肝炎表面抗原之組分 IV ： a). 將磷酸鋁轉移到容器/器皿中 b). 添加乙型肝炎表面抗原 c). 用醋酸/氫氧化鈉將pH調節至4.5至5.5 d). 等待穩定 e). 用氫氧化鈉/碳酸鈉將pH調節至5.5至6.5 f). 等待穩定4. 製造包含 D 、 T 、 wP 、 HBsAg 、 Hib PRP-TT 接合物、 IPV 及防腐劑之聯合疫苗組合物的方法 1.將生理鹽水添加到攪拌器皿/容器中； 2.添加組分I 3.將組分II在組分I中混合並且在RT下攪拌30-45分鐘。 4.在上述混合物中添加組分III，然後在RT下攪拌30-60分鐘。 5.將組分IV加入步驟4中獲得之混合物中，然後在RT下攪拌30-60分鐘。 6.將組分V加入步驟5中獲得之混合物中，然後在6–16℃下攪拌30-60分鐘。 7.將組分VI加入步驟6中獲得之混合物中，然後在6-16℃下攪拌。 8.在6-16℃下，將以下揭示之防腐劑組合中之一種加入步驟7中獲得之混合物中。 a)2-苯氧基乙醇，其量為約1 mg/0.5 ml至6 mg/0.5 ml(v/v)；或 b)2-苯氧基乙醇，其量為約1 mg/0.5 ml至6 mg/0.5 ml(v/v)及對羥基苯甲酸甲酯，其使用濃度為0.1-1.5 mg/0.5 ml(w/v)；或 c)2-苯氧基乙醇，其量為約1 mg/0.5 ml至6 mg/0.5 ml(v/v)及對羥基苯甲酸丙酯，其使用濃度為0.05-0.2 mg/0.5 ml(w/v)；或 d)2-苯氧基乙醇，其量為約1 mg/0.5 ml至6 mg/0.5 ml(v/v)，對羥基苯甲酸甲酯，其使用濃度為0.1-1.5 mg/0.5 ml(w/v)及對羥基苯甲酸丙酯，其使用濃度為0.05-0.2 mg/0.5 ml(w/v)。 9.檢查pH，如果需要，用氫氧化鈉/碳酸鈉將pH調節至6.0至7.5 10.用鹽水(0.9%)補足在步驟9中得到之體積，隨後攪拌3小時。實例 6 ：抗原之吸附、效力及穩定性

觀察結果： •用半劑量濃度之IPV製造之六價疫苗批次已顯示出令人鼓舞之測試結果。 •發現在半濃度IPV下製造之六價疫苗之IPV體內功效與具有全劑量IPV之現有疫苗(SIIPL製造之Poliovac)相當。實例 7 ：抗菌能力測試

Example 5 : Method of manufacturing a combined vaccine This example briefly introduces the manufacturing method of a combined vaccine composition containing D , T , wP , HBsAg , Hib PRP-TT conjugate, IPV and preservative: Component I- Diphtheria adsorbed by alum Toxoid component II- tetanus toxoid component III-wP antigen adsorbed by alum ( as disclosed in Example 3 ) Component IV- hepatitis B surface antigen component V-Hib PRP conjugate ( such as Disclosed in Example 2 ) Component VI-IPV antigen ( as disclosed in Example 4 ) 1. Prepare component I containing diphtheria toxoid adsorbed by alum : a). Transfer aluminum phosphate to a container/vessel b) Add diphtheria toxoid c). Adjust pH to 4.5 to 5.5 with acetic acid/sodium hydroxide d). Wait for stabilization e). Adjust pH to 5.5 to 6.5 with sodium hydroxide/sodium carbonate f). Wait for stabilization 2. Prepare component II containing tetanus toxoid adsorbed by alum : a). Transfer aluminum phosphate to a container/ware b). Add tetanus toxoid c). Adjust pH to 4.5 to 5.5 with acetic acid/sodium hydroxide d ). Wait for stabilization e). Adjust pH to 5.5 to 6.5 with sodium hydroxide/sodium carbonate f). Wait for stabilization 3. Prepare component IV containing hepatitis B surface antigen adsorbed by alum : a). Transfer to a container/ware b). Add hepatitis B surface antigen c). Adjust pH to 4.5 to 5.5 with acetic acid/sodium hydroxide d). Wait for stability e). Adjust pH to pH with sodium hydroxide/sodium carbonate 5.5 to 6.5 f). Waiting for stabilization 4. Method of manufacturing a combined vaccine composition containing D , T , wP , HBsAg , Hib PRP-TT conjugate, IPV and preservatives 1. Add physiological saline to the mixing vessel/container 2. Add component I 3. Mix component II in component I and stir at RT for 30-45 minutes. 4. Add component III to the above mixture, and then stir at RT for 30-60 minutes. 5. Add component IV to the mixture obtained in step 4, and then stir at RT for 30-60 minutes. 6. Add component V to the mixture obtained in step 5, and then stir at 6-16°C for 30-60 minutes. 7. Add component VI to the mixture obtained in step 6, and then stir at 6-16°C. 8. At 6-16°C, add one of the preservative combinations disclosed below to the mixture obtained in step 7. a) 2-phenoxyethanol, whose amount is about 1 mg/0.5 ml to 6 mg/0.5 ml (v/v); or b) 2-phenoxyethanol, whose amount is about 1 mg/0.5 ml to 6 mg/0.5 ml (v/v) and methyl p-hydroxybenzoate, the use concentration is 0.1-1.5 mg/0.5 ml (w/v); or c) 2-phenoxyethanol, the amount is about 1 mg/0.5 ml to 6 mg/0.5 ml (v/v) and propyl p-hydroxybenzoate at a concentration of 0.05-0.2 mg/0.5 ml (w/v); or d) 2-phenoxyethanol, The amount is about 1 mg/0.5 ml to 6 mg/0.5 ml (v/v), methyl paraben, and its use concentration is 0.1-1.5 mg/0.5 ml (w/v) and propyl paraben Ester, the use concentration is 0.05-0.2 mg/0.5 ml(w/v). 9. Check the pH, if necessary, adjust the pH to 6.0 to 7.5 with sodium hydroxide/sodium carbonate. 10. Make up the volume obtained in step 9 with brine (0.9%), and then stir for 3 hours. Example 6 : Adsorption, potency and stability of antigen

Observations: • The hexavalent vaccine batch manufactured with half-dose IPV has shown encouraging test results. • It is found that the IPV in vivo efficacy of the hexavalent vaccine manufactured under half-concentration IPV is equivalent to the existing vaccine with full dose IPV (Poliovac manufactured by SIIPL). Example 7 : Antibacterial ability test

While developing a multi-dose combination vaccine containing D, T, wP, Hib, HBsAg and IPV vaccines, the inventors have first added 2-phenoxyethanol (2-PE) at a dose of 2.5 mg/0.5 ml. ) Has tested its antibacterial ability, and 2-phenoxyethanol is usually used as a preservative in this technology. However, in the combination vaccine based on DPT, it was found that 2-PE has lower antibacterial activity against yeast and fungi than thimerosal.

Increasing the amount of 2-PE (preservative) in order to meet the required standards may increase the safety of young children who are vaccinated subjects and may also affect the stability of the final product. Furthermore, the amount of preservatives contained in the vaccine should meet the requirements defined in the U.S. Pharmacopoeia, European Pharmacopoeia, WHO Pharmacopoeia or a combination of vaccine safety.

抗菌能力之篩選： In this regard, the inventors conducted experiments to develop a novel composition by combining 2-PE with other preservatives such as para-hydroxybenzoic acid in a multi-dose combination vaccine meeting safety and antibacterial ability standards. The ester combination meets the requirements for antibacterial ability. In the present invention, the antibacterial ability test is performed according to the European Pharmacopoeia Class B (EP-B) standard required by WHO for vaccine products.

Screening of antibacterial ability:

NA- 不可用； 0.5% 2PE – 2.5 mg/0.5 ml 劑量； 0.4% 2PE – 2 mg/0.5 ml 劑量； 0.18%MP – 0.9 mg/0.5 ml 劑量； 0.02%PP – 0.1mg/0.5 ml 劑量； CFU- 菌落形成單位 觀察結果： •已觀察到，按照歐洲藥典B類，發現以不同組合製造之所有六價疫苗均符合防腐功效。但是，發現使用不同組合時，其功效有所不同。 •與其他防腐劑組合(即僅2PE、2PE與PP、2PE與MP及PP與MP)相比，含有2PE、MP及PP之六價疫苗之防腐功效係非常有效的。 •亦應注意，與含有0.4%2PE之相同組合相比，發現含有0.5%2PE及PP與MP之六價疫苗之防腐功效係更有效的。實例 8 ： SIIPL 之減劑量聯合疫苗與 Easy Six(Panacea) At the 0th hour, the hexavalent combined vaccine preparation disclosed in Example 1 was inoculated with a total of six microorganisms, including four different bacteria-Staphylococcus aureus (ATCC NO.-6538), Pseudomonas aeruginosa (ATCC) NO.-9027), Escherichia coli (ATCC NO.-8739) and Staphylococcus aureus (environmental isolate EMI); a yeast-Candida albicans (ATCC NO.-10231) and a fungus-Aspergillus brasiliensis (ATCC NO. -16404), added to the vaccine formulation in an amount of 105 to 106 CFU/mL. Then, samples of bacteria, fungi, and yeast were collected on the 0th hour, 24th hour, 7th day, 14th day, and 28th day, and cultured in a solid medium. The number of colonies was counted between the 3rd and 5th days, and Calculate the log reduction of the colony. The results are shown in Table 38 below.

NA- not available; 0.5% 2PE – 2.5 mg/0.5 ml dose; 0.4% 2PE – 2 mg/0.5 ml dose; 0.18%MP – 0.9 mg/0.5 ml dose; 0.02%PP – 0.1 mg/0.5 ml dose; CFU - colony forming units observations: • it has been observed, according to the European Pharmacopoeia class B, we found that all of the hexavalent vaccine manufactured in different combinations are in line with anti-corrosion effect. However, it was found that when different combinations were used, their efficacy was different. • Compared with other preservative combinations (ie only 2PE, 2PE and PP, 2PE and MP, and PP and MP), the antiseptic effect of the hexavalent vaccine containing 2PE, MP and PP is very effective. • It should also be noted that compared with the same combination containing 0.4% 2PE, the antiseptic effect of the hexavalent vaccine containing 0.5% 2PE and PP and MP was found to be more effective. Example 8 : Reduced- dose combination vaccine of SIIPL and Easy Six (Panacea)

Claims (61)

An all-liquid multi-dose immunogenic composition comprising: (i) Diphtheria toxoid, (D); (ii) Tetanus toxoid, (T); (iii) Inactivated whole-cell pertussis, (wP) or acellular pertussis, (aP); (iv) Hepatitis B virus surface antigen, (HBsAg); (v) Haemophilus influenzae type b antigen, (Hib); (vi) Inactivated poliovirus antigen, (IPV), in which the amount of IPV type 1 is 1-50 DU/0.5 ml, and the amount of IPV type 3 is 1-50 DU/0.5 ml; and (vii) The combination of 2-phenoxyethanol and at least one other preservative. For example, the immunogenic composition of the first item of the scope of patent application, wherein the ratio of the inactivated whole-cell pertussis strains in the composition is 1:1:0.25:0.25 with Bordetella pertussis strains 134, 509, 25525 and 6229 . For example, the immunogenic composition of the first item of the patent application, wherein the acellular pertussis antigen includes a modified adenylate cyclase, pertussis toxoid (PT), filamentous hemagglutinin (FHA), and Bacillus pertussis One or more antigens of Adhesin (P69 or PRN) or Fimbrin (FIM 1, 2 and 3). For example, the immunogenic composition of the first item of the patent application, wherein the Hib antigen is bonded to the Hib polyribosyl ribitol phosphate (PRP) polysaccharide of the carrier protein using cyanation bonding chemistry or reductive amination bonding chemistry, wherein the The cyanating reagent is selected from cyanogen bromide, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), 1-cyano-4-pyrrolidinylpyridine tetrafluoroborate (CPPT), 1-cyanoimidazole (1-CI), 1-cyanobenzene and triazole (1-CBT) or 2-cyanotakoujing-3(2H)one (2-CPO); and the carrier protein is selected from The following groups: tetanus toxoid, CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, tetanus toxoid fragment C, pertussis toxoid, Haemophilus influenzae protein D, Escherichia coli LT, large intestine Bacillus ST and Pseudomonas aeruginosa exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, Bacillus anthracis protective antigen (PA) and Bacillus anthracis detoxification edema factor (EF) and lethal factor (LF), egg Albumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptide, heat shock protein, pertussis protein, cytokine , Lymphatic factor, hormone, growth factor, artificial protein containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N19, iron uptake protein, protein from Clostridium difficile and Streptococcus agalactiae with or without linker Toxin A or B. Such as the immunogenic composition of item 1 in the scope of the patent application, wherein the composition also contains IPV type 2 in an amount of 1-20 DU/0.5ml. For example, the immunogenic composition of items 1 and 5 of the scope of patent application, wherein the IPV type 1 is a Mahoney strain or a Sabin strain; and/or wherein the IPV type 2 is a MEF-1 strain or a Sabin strain; and/or wherein IPV type 3 is Saukett strain or Sabin strain. For example, the immunogenic composition of item 1 of the scope of the patent application, wherein the other preservative is selected from the group consisting of Phemerol chloride, phenol, m-cresol, formaldehyde, aniline chloride, benzyl alcohol, and chlorobutanol , P-chlorom-cresol, benzyl alcohol and parabens selected from the group of methyl paraben, ethyl paraben, propyl paraben and butyl paraben Group. For example, the immunogenic composition of item 1 of the scope of patent application, wherein the composition contains 2-phenoxyethanol in an amount of 1-6 mg/0.5ml(v/v); 0.1-1.5 mg/0.5ml(w /v) of methyl parahydroxybenzoate and 0.05-0.2 mg/0.5ml(w/v) of propyl parahydroxybenzoate. For example, the immunogenic composition of item 1 in the scope of the patent application, wherein the D, T, wP, HBsAg, Hib and IPV are not adsorbed on the adjuvant. For example, the immunogenic composition of item 1 in the scope of the patent application, wherein the D, T and HBsAg are individually adsorbed on aluminum salts (Al ³⁺ ) such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposome or oil-in-water emulsion adjuvant. For example, the immunogenic composition of item 10 in the scope of patent application, wherein the D, T, and HBsAg are separately adsorbed on aluminum phosphate (AlPO ₄ ) adjuvant. Such as the immunogenic composition of item 10 in the scope of the patent application, wherein the D antigen is adsorbed on the aluminum salt with an adsorption percentage of at least 50%. Such as the immunogenic composition of item 10 in the scope of the patent application, wherein the T antigen is adsorbed on the aluminum salt with an adsorption percentage of at least 40%. Such as the immunogenic composition of item 10 in the scope of the patent application, wherein the HBsAg antigen is adsorbed on the aluminum salt with an adsorption percentage of at least 50%. For example, the immunogenic composition of items 1, 5 and 6 of the scope of patent application, wherein the IPV antigen is adsorbed on the aluminum salt of hydroxide (Al(OH) ₃ ) or phosphate (AlPO ₄ ), IPV type 1 The adsorption percentage is in the range of 10-100%, the IPV 2 type is in the range of 10-100%, and the IPV 3 type is in the range of 10-100%. Such as the immunogenic composition of the first item in the scope of patent application, wherein the adsorption percentage of Hib antigen on any adjuvant is less than 20%. For example, the immunogenic composition of any one of items 1 to 16 in the scope of patent application, wherein the total aluminum content (Al ³⁺ ) in the composition is 0.1 to 0.6 mg/0.5 ml. Such as the immunogenic composition of item 1 in the scope of the patent application, wherein the composition further comprises a buffer selected from sodium chloride or phosphate buffered saline. Such as the immunogenic composition of the 18th patent application, wherein the composition contains sodium chloride at a concentration of 0.5% to 1.5% as a buffer. Such as the immunogenic composition of item 1 in the scope of the patent application, wherein the composition further comprises a pharmaceutically acceptable transporter, excipient, binder, carrier, isotonic agent, emulsifier or wetting agent. Such as the immunogenic composition of claim 20, wherein the composition comprises a pharmaceutically acceptable excipient selected from the group of sugars, polyols, surfactants, polymers, salts, amino acids or pH regulators Shape agent. For example, the immunogenic composition of any one of items 1 to 21 in the scope of patent application, wherein the single-dose immunogenic composition does not contain preservatives. For example, the immunogenic composition of item 1 of the scope of patent application, wherein the composition further comprises one or more antigens selected from the following group: Haemophilus influenzae (a, c, d, e, f serotypes and Encapsulated strains), hepatitis (A, C, D, E, F and G strains), rotavirus, Neisseria meningitidis A antigen, Neisseria meningitidis C antigen, Neisseria meningitidis W -135 antigen, Neisseria meningitidis Y antigen, Neisseria meningitidis X antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis B antigen, Staphylococcus aureus antigen, anthrax, BCG, human papilloma virus , S. typhi antigen, S. typhi antigen, modified adenylate cyclase, malaria antigen (RTS, S), measles, mumps, rubella, flavivirus antigen, dengue fever, Zika, Ebola , Qu Gong, Japanese encephalitis and diarrhea antigens. For example, the immunogenic composition of any one of items 1 to 23 in the scope of the patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 Lf to 10 Lf of T antigen; About 12 to 16 IOU of wP antigen; 7 to 15 µg of HBsAg; 7 to 13 µg of Hib antigen; IPV antigen, respectively 1-50 DU amount of type 1 (Mahoney strain or Sabin strain ) And type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-benzene in the amount of 1 to 6 mg (v/v) Oxyethanol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-50 DU, Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-20 DU and type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content in the amount of 0.1 to 0.6 mg (Al ³⁺ ); about 1 to 6 mg (v/v) of 2-phenoxyethanol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-50 DU, and Type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-phenoxy in the amount of 1 to 6 mg (v/v) Ethanol; 0.05-0.2 mg (w/v) of propyl paraben. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-50 DU, Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-20 DU and type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content in the amount of 0.1 to 0.6 mg (Al ³⁺ ); 2-phenoxyethanol in the amount of 1 to 6 mg (v/v); propyl p-hydroxybenzoate in the amount of 0.05-0.2 mg (w/v). For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-50 DU, Type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-phenoxy in the amount of 1 to 6 mg (v/v) Alcohol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate; 0.05-0.2 mg (w/v) of propyl p-hydroxybenzoate. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-50 DU, Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-20 DU and type 3 (Saukett strain or Sabin strain) in the amount of 1-50 DU; total aluminum content in the amount of 0.1 to 0.6 mg (Al ³⁺ ); 2-phenoxyethanol in the amount of 1 to 6 mg (v/v); methyl parahydroxybenzoate in the amount of 0.1-1.5 mg (w/v); 0.05-0.2 mg (w/v) The amount of propyl paraben. For example, the immunogenic composition of any one of items 1 to 23 in the scope of the patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 Lf to 10 Lf of T antigen; 12 to 16 IOU of wP antigen; 7 to 15 µg of HBsAg; 7 to 13 µg of Hib antigen; IPV antigen, respectively 1-25 DU of type 1 (Mahoney strain or Sabin strain) Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-benzene in the amount of 1 to 6 mg (v/v) Oxyethanol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain), respectively, in the amount of 1-25 DU, Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-10 DU and Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg ); about 1 to 6 mg (v/v) of 2-phenoxyethanol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain), respectively, in the amount of 1-25 DU, Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-phenoxy in the amount of 1 to 6 mg (v/v) Alcohol; 0.05-0.2 mg (w/v) of propyl paraben. For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain), respectively, in the amount of 1-25 DU, Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-10 DU and Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg ); 2-phenoxyethanol in the amount of 1 to 6 mg (v/v); propyl p-hydroxybenzoate in the amount of 0.05-0.2 mg (w/v). For example, the immunogenic composition of any one of items 1 to 23 in the scope of patent application, wherein 0.5 ml of the composition contains: 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 WP antigen in the amount of up to 16 IOU; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain), respectively, in the amount of 1-25 DU, Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; 2-phenoxy in the amount of 1 to 6 mg (v/v) Alcohol; 0.1-1.5 mg (w/v) of methyl p-hydroxybenzoate; 0.05-0.2 mg (w/v) of propyl p-hydroxybenzoate. Such as the immunogenic composition of any one of items 1 to 23 in the scope of the patent application, wherein 0.5 ml of the composition contains 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 to 16 IOU amount of wP antigen; 7 to 15 µg amount of HBsAg; 7 to 13 µg amount of Hib antigen; IPV antigen, respectively, 1-25 DU amount of type 1 (Mahoney strain or Sabin strain), Type 2 (MEF-1 strain or Sabin strain) in the amount of 1-10 DU and Type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg ); 2-phenoxyethanol in the amount of 1 to 6 mg (v/v); methyl parahydroxybenzoate in the amount of 0.1-1.5 mg (w/v); 0.05-0.2 mg (w/v) The amount of propyl paraben. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain) and 32 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl paraben in the amount of 0.9 mg (w/v) Ester; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, respectively 40 DU The amount of type 1 (Mahoney strain or Sabin strain), and the amount of 32 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP Antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain) and 32 DU of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl p-hydroxybenzoate in the amount of 0.9 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain) Or Sabin strain) and type 3 (Saukett strain or Sabin strain) in the amount of 32 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy in the amount of 2.5 mg (v/v) Ethanol; 0.9 mg (w/v) of methyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg 10 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain or Sabin strain), and 32 DU Type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) of 2-phenoxyethanol; 0.9 mg (w/v) The amount of methylparaben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; The IPV antigens are 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain or Sabin strain), and 32 DU of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl p-hydroxybenzoate in the amount of 0.9 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain) and 32 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al3+) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); propyl paraben in the amount of 0.1 mg (w/v); Or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, each of which is 40 DU Type 1 (Mahoney strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 32 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) 2-Phenoxyethanol; 0.1 mg (w/v) of propyl paraben. 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen, each of which is 1 of 40 DU Type (Mahoney strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 32 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2 of the amount of 2.5 mg (v/v) -Phenoxyethanol; 0.1 mg (w/v) of propyl paraben. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain) Or Sabin strain) and type 3 (Saukett strain or Sabin strain) in the amount of 32 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy in the amount of 2.5 mg (v/v) Ethanol; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg 10 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain or Sabin strain), and 32 DU Type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) 2-phenoxyethanol; 0.1 mg (w/v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; The IPV antigens are 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain or Sabin strain), and 32 DU of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); propyl paraben in the amount of 0.1 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain) and 32 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl paraben in the amount of 0.9 mg (w/v) Ester; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg ; 10 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain) and 32 DU of type 3 (Saukett strain or Sabin strain); no more than 0.55 mg Total aluminum content (Al ³⁺ ); 2.5 mg(v/v) of 2-phenoxyethanol; 0.9 mg(w/v) of methyl parahydroxybenzoate; 0.1 mg(w/v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigens: 40 DU type 1 (Mahoney strain or Sabin strain) and 32 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg(v/v) 2-phenoxyethanol; 0.9 mg(w/v) methyl paraben; 0.1 mg(w/v) propyl paraben . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain) Or Sabin strain) and type 3 (Saukett strain or Sabin strain) in the amount of 32 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy in the amount of 2.5 mg (v/v) Ethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU amount of wP antigen; 15 µg amount of HBsAg; 10 µg amount of Hib antigen; IPV antigen, respectively 40 DU amount of type 1 (Mahoney strain or Sabin strain), 8 DU amount Type 2 (MEF-1 strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 32 DU; total aluminum content (Al3+) not more than 0.55 mg; 2.5 mg (v/v) 2-Phenoxyethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 25 Lf of D antigen ; 10 Lf amount of T antigen; 16 IOU amount of wP antigen; 15 µg amount of HBsAg; 13 µg amount of Hib antigen; IPV antigen, respectively 40 DU amount of type 1 (Mahoney strain or Sabin strain ), 8 DU type 2 (MEF-1 strain or Sabin strain) and 32 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg(v/v) 2-phenoxyethanol; 0.9 mg(w/v) methyl parahydroxybenzoate; 0.1 mg(w/v) propyl paraben. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain) and 16 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl paraben in the amount of 0.9 mg (w/v) Ester; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, respectively, 20 DU The amount of type 1 (Mahoney strain or Sabin strain), and the amount of 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP Antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain) and 16 DU of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl p-hydroxybenzoate in the amount of 0.9 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain), 4 DU of type 2 (MEF-1 strain) Or Sabin strain) and 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy group in the amount of 2.5 mg (v/v) Ethanol; 0.9 mg (w/v) of methyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg ; 10 µg amount of Hib antigen; IPV antigen, respectively 20 DU amount of type 1 (Mahoney strain or Sabin strain), 4 DU amount of type 2 (MEF-1 strain or Sabin strain) and 16 DU amount Type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) of 2-phenoxyethanol; 0.9 mg (w/v) The amount of methylparaben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; The IPV antigens are type 1 (Mahoney strain or Sabin strain) for 20 DU, type 2 (MEF-1 strain or Sabin strain) for 4 DU, and type 3 (Saukett strain or Sabin strain for 16 DU) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl p-hydroxybenzoate in the amount of 0.9 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain) and 16 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propyl paraben in an amount of 0.1 mg (w/v) Ester; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, respectively, 20 DU The amount of type 1 (Mahoney strain or Sabin strain), and the amount of 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.1 mg (w/v) of propyl paraben. 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen, each of which is 1 of 20 DU Type (Mahoney strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 16 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) amount 2 -Phenoxyethanol; 0.1 mg (w/v) of propyl paraben. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain), 4 DU of type 2 (MEF-1 strain) Or Sabin strain) and 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy group in the amount of 2.5 mg (v/v) Ethanol; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg ; 10 µg amount of Hib antigen; IPV antigen, respectively 20 DU amount of type 1 (Mahoney strain or Sabin strain), 4 DU amount of type 2 (MEF-1 strain or Sabin strain) and 16 DU amount Type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) 2-phenoxyethanol; 0.1 mg (w/v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; The IPV antigens are type 1 (Mahoney strain or Sabin strain) for 20 DU, type 2 (MEF-1 strain or Sabin strain) for 4 DU, and type 3 (Saukett strain or Sabin strain for 16 DU) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); propyl paraben in the amount of 0.1 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain), and 16 DU of type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); p-hydroxybenzoic acid in the amount of 0.9 mg (w/v) Methyl ester; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain), and 16 DU of type 3 (Saukett strain or Sabin strain); no more than 0.55 mg of total aluminum content (Al ³⁺ ); 2.5 mg(v/v) of 2-phenoxyethanol; 0.9 mg(w/v) of methyl parahydroxybenzoate; 0.1 mg(w/ v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib Antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain) and 16 DU of type 3 (Saukett strain or Sabin strain); total aluminum content of not more than 0.55 mg (Al ³⁺ ); 2.5 mg (v/v) of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of parahydroxybenzoic acid Propyl ester. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 20 DU of type 1 (Mahoney strain or Sabin strain), 4 DU of type 2 (MEF-1 strain) Or Sabin strain) and 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy group in the amount of 2.5 mg (v/v) Ethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU amount of wP antigen; 15 µg amount of HBsAg; 10 µg amount of Hib antigen; IPV antigen, respectively 20 DU amount of type 1 (Mahoney strain or Sabin strain), 4 DU amount Type 2 (MEF-1 strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 16 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 25 Lf of D antigen; 10 Lf amount of T antigen; 16 IOU amount of wP antigen; 15 µg amount of HBsAg; 13 µg amount of Hib antigen; IPV antigen, respectively 20 DU amount of type 1 (Mahoney strain or Sabin strain), 4 DU type 2 (MEF-1 strain or Sabin strain) and 16 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg ; 2.5 mg(v/v) 2-phenoxyethanol; 0.9 mg(w/v) methyl paraben; 0.1 mg(w/v) methyl paraben ester. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, respectively 7.5 DU of type 1 (Mahoney strain or Sabin strain), and 6 DU of type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); p-hydroxybenzoic acid in the amount of 0.9 mg (w/v) Methyl ester; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, respectively 7.5 DU The amount of type 1 (Mahoney strain or Sabin strain) and 6 DU of type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP Antigen; 15 µg amount of HBsAg; 13 µg amount of Hib antigen; IPV antigen, respectively 7.5 DU amount of type 1 (Mahoney strain or Sabin strain) and 6 DU amount of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); methyl p-hydroxybenzoate in the amount of 0.9 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 7.5 DU of type 1 (Mahoney strain or Sabin strain), 1.5 DU of type 2 (MEF-1 strain) Or Sabin strain) and 6 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al3+) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); 0.9 mg (w/v) of methyl parahydroxybenzoate; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 The amount of Hib antigen in µg; the IPV antigen, which is 7.5 DU of type 1 (Mahoney strain or Sabin strain), 1.5 DU of type 2 (MEF-1 strain or Sabin strain), and 3 of 6 DU. Type (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) 2-phenoxyethanol; 0.9 mg (w/v) Or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen Type 1 (Mahoney strain or Sabin strain) with an amount of 7.5 DU, Type 2 (MEF-1 strain or Sabin strain) with an amount of 1.5 DU, and Type 3 (Saukett strain or Sabin strain) with an amount of 6 DU; Total aluminum content (Al ³⁺ ) of not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); and methyl parahydroxybenzoate in the amount of 0.9 mg (w/v). The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 7.5 DU of type 1 (Mahoney strain or Sabin strain) and 6 DU of type 3 (Saukett strain or Sabin) Strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propyl paraben in an amount of 0.1 mg (w/v) Ester; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, respectively 7.5 DU The amount of type 1 (Mahoney strain or Sabin strain) and 6 DU of type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.1 mg (w/v) of propyl paraben. 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; IPV antigen, each of which is 1 of 7.5 DU Type (Mahoney strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 6 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2 of the amount of 2.5 mg (v/v) -Phenoxyethanol; 0.1 mg (w/v) of propyl paraben. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 7.5 DU of type 1 (Mahoney strain or Sabin strain), 1.5 DU of type 2 (MEF-1 strain) Or Sabin strain) and 6 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy group in the amount of 2.5 mg (v/v) Ethanol; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg ; 10 µg amount of Hib antigen; IPV antigen, respectively 7.5 DU amount of type 1 (Mahoney strain or Sabin strain), 1.5 DU amount of type 2 (MEF-1 strain or Sabin strain) and 6 DU amount Type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) 2-phenoxyethanol; 0.1 mg (w/v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib antigen; The IPV antigens are 40 DU of type 1 (Mahoney strain or Sabin strain), 8 DU of type 2 (MEF-1 strain or Sabin strain), and 32 DU of type 3 (Saukett strain or Sabin strain) ); Total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); propyl paraben in the amount of 0.1 mg (w/v) . The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, respectively 7.5 DU of type 1 (Mahoney strain or Sabin strain), and 6 DU of type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in the amount of 2.5 mg (v/v); p-hydroxybenzoic acid in the amount of 0.9 mg (w/v) Methyl ester; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU of wP antigen; 15 µg of HBsAg; 10 µg of Hib antigen; IPV antigen, 7.5 DU of type 1 (Mahoney strain or Sabin strain), and 6 DU of type 3 (Saukett strain or Sabin strain); no more than 0.55 mg of total aluminum content (Al ³⁺ ); 2.5 mg(v/v) of 2-phenoxyethanol; 0.9 mg(w/v) of methyl parahydroxybenzoate; 0.1 mg(w/ v) The amount of propyl paraben; or 25 Lf of D antigen; 10 Lf of T antigen; 16 IOU of wP antigen; 15 µg of HBsAg; 13 µg of Hib Antigen; IPV antigen, type 1 (Mahoney strain or Sabin strain) and 6 DU type 3 (Saukett strain or Sabin strain) in the amount of 7.5 DU respectively; total aluminum content of not more than 0.55 mg (Al ³⁺ ); 2.5 mg (v/v) of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of parahydroxybenzoic acid Propyl ester. The immunogenic composition of any one of the aforementioned patent applications, wherein 0.5 ml of the composition contains any one of the following: 10 Lf of D antigen; 2 Lf of T antigen; 12 IOU of wP antigen; 8 µg of HBsAg; 8 µg of Hib antigen; IPV antigen, 7.5 DU of type 1 (Mahoney strain or Sabin strain), 1.5 DU of type 2 (MEF-1 strain) Or Sabin strain) and 6 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxy group in the amount of 2.5 mg (v/v) Ethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 20 Lf of D antigen; 4 Lf of T antigen; 14 IOU amount of wP antigen; 15 µg amount of HBsAg; 10 µg amount of Hib antigen; IPV antigen, respectively 7.5 DU amount of type 1 (Mahoney strain or Sabin strain), 1.5 DU amount Type 2 (MEF-1 strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) with an amount of 6 DU; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2.5 mg (v/v) The amount of 2-phenoxyethanol; 0.9 mg (w/v) of methyl paraben; 0.1 mg (w/v) of propyl paraben; or 25 Lf of D antigen; 10 Lf amount of T antigen; 16 IOU amount of wP antigen; 15 µg amount of HBsAg; 13 µg amount of Hib antigen; IPV antigen, respectively 7.5 DU amount of type 1 (Mahoney strain or Sabin strain), 1.5 DU type 2 (MEF-1 strain or Sabin strain) and 6 DU type 3 (Saukett strain or Sabin strain); total aluminum content (Al ³⁺ ) not more than 0.55 mg ; 2.5 mg(v/v) 2-phenoxyethanol; 0.9 mg(w/v) methyl paraben; 0.1 mg(w/v) methyl paraben ester. A method of manufacturing a full liquid multi-dose immunogenic composition, the composition comprising: (i) Diphtheria toxoid, (D); (ii) Tetanus toxoid, (T); (iii) Inactivated whole-cell pertussis, (wP) or acellular pertussis, (aP); (iv) Hepatitis B virus surface antigen, (HBsAg); (v) Haemophilus influenzae type b antigen, (Hib); (vi) Inactivated poliovirus antigen, (IPV) and (vii) A combination of 2-phenoxyethanol and at least one paraben; The method includes the following steps: a). Add physiological saline to a stirring vessel/container; b). Add component-I containing diphtheria toxoid; c). Add component-II containing tetanus toxoid; d). Add the component-Ⅲ containing inactivated whole-cell pertussis antigen or acellular pertussis (aP); e). Add component-IV containing hepatitis B surface antigen; f). Add component-V containing Hib antigen; g). Add the component-VI containing IPV antigen, where the amount of IPV type 1 (Mahoney strain or Sabin strain) is 1-50 DU/0.5 ml; and the amount of IPV type 3 (Saukett strain or Sabin strain) is 1 -50 DU/0.5 ml; h). Add a combination of preservatives selected from the following: 1 to 6 mg/0.5 ml (v/v) of 2-phenoxyethanol and 0.1 to 1.5 mg/0.5 ml (w/v) of methyl parahydroxybenzoate; or 1 to 6 mg/0.5 ml (v/v) of 2-phenoxyethanol and 0.05 to 0.2 mg/0.5 ml (w/v) of propyl paraben; or 1 to 6 mg/0.5 ml(v/v) of 2-phenoxyethanol, 0.1 to 1.5 mg/0.5 ml(w/v) of methyl paraben and 0.05 to 0.2 mg/0.5 ml (w/v) of propyl paraben; i). Adjust the pH to 6.0 to 7.0 with sodium hydroxide/sodium carbonate; and j). Add saline to make up the volume. Such as the method of item 54 in the scope of the patent application, wherein the component-VI further comprises 1-20 DU/0.5 ml of IPV antigen type 2 (MEF-1 strain or Sabin strain). For example, the 54th method in the scope of patent application, wherein the preparation of component I includes the following steps: a). Transfer aluminum phosphate to the container/ware; b). Add diphtheria toxoid; c). Adjust the pH to 4.5 to 5.5 with acetic acid/sodium hydroxide; d). Stable; e). Adjust the pH to 5.5 to 6.5 with sodium hydroxide/sodium carbonate; and f). Stabilize with histidine buffer. For example, the 54th method in the scope of patent application, wherein the preparation of the component II includes the following steps: a). Transfer aluminum phosphate to the container/ware; b). Add tetanus toxoid; c). Adjust the pH to 4.5 to 5.5 with acetic acid/sodium hydroxide; d). Stable; e). Adjust the pH to 5.5 to 6.5 with sodium hydroxide/sodium carbonate; and f). Stabilize with histidine buffer. For example, the 54th method in the scope of patent application, wherein the preparation of component II includes the following steps: a). Bordetella pertussis strain 134 is inactivated at 56°C for 10-15 minutes in the presence of formaldehyde; b). Bordetella pertussis strain 509 is inactivated at 56°C for 10-15 minutes in the presence of formaldehyde; c). Bordetella pertussis strains 25525 and 6229 are inactivated at 56°C for 10-15 minutes in the presence of formaldehyde; c) Bordetella pertussis strain 6229 is inactivated at 56°C for 10-15 minutes in the presence of formaldehyde; d). Then mix and inactivate the Bordetella pertussis strains 134, 509, 25525 and 6229 at a ratio of 1:1:0.25:0.25; e). Adsorbed on aluminum-based adjuvant as appropriate; The method does not contain thimerosal and the inactivated whole-cell pertussis antigen remains non-agglomerated and homogeneous, which results in reduced reactogenicity and provides better efficacy over a longer period of time. For example, the method of item 54 in the scope of patent application, wherein the preparation of the component IV includes the following steps: a). Transfer aluminum phosphate to the container/ware; b). Add hepatitis B surface antigen; c). Adjust the pH to 4.5 to 5.5 with acetic acid/sodium hydroxide; d). Stable; e). Adjust the pH to 5.8 to 6.8 with sodium hydroxide/sodium carbonate; and f). Stable. Such as the method of item 54 in the scope of patent application, wherein the preparation of component-V includes the following steps: a). Fermentation of Haemophilus influenzae type b; b). Inactivated at 37°C for 2 hours in the presence of 0.1% formaldehyde; c). Purify Hib phosphate polyribosyl ribitol (PRP) polysaccharide; d) In the presence of adipic acid dihydrazine (ADH) linker, the purified product of step c is joined with tetanus toxoid (TT) using bromide cyanocyanation joining chemistry; e). Purify the conjugate of step d; and f). The purified conjugate is preferably filtered through a 0.22 µm filter; The percentage of free PRP in the whole purified Hib conjugate does not exceed 5%. A whole liquid multi-dose immunogenic composition, wherein 0.5 ml of the composition contains 10 to 25 Lf of D antigen; 2 to 10 Lf of T antigen; 12 to 16 IOU of wP antigen; HBsAg in the amount of 7 to 15 µg; Hib antigen in the amount of 7 to 13 µg; IPV antigen, type 1 (Mahoney strain or Sabin strain) in the amount of 1-25 DU, and type 2 in the amount of 1-10 DU (MEF-1 strain or Sabin strain) and type 3 (Saukett strain or Sabin strain) in the amount of 1-20 DU; total aluminum content (Al ³⁺ ) in the amount of 0.1 to 0.6 mg; about 1 to 6 mg (v /v) 2-phenoxyethanol; wherein the composition is prepared by the following method: a). Adding physiological saline to a stirring vessel/container; b). Adding components containing diphtheria toxoid- I; c). Add tetanus toxoid-containing component-II; d). Add inactivated whole-cell pertussis antigen or acellular pertussis (aP) component-Ⅲ; e). Add hepatitis B surface antigen Component-IV; f). Add component-V containing Hib antigen; g). Add component-VI containing IPV antigen, where the amount of IPV type 1 (Mahoney strain or Sabin strain) is 1-25 DU ; The amount of type 2 (MEF-1 strain or Sabin strain) is 1-10 DU and the amount of type 3 (Saukett strain or Sabin strain) is 1-20 DU; h). Add 1 to 6 mg/0.5 ml (v /v) 2-phenoxyethanol i). Adjust the pH to 6.0 to 7.0 with sodium hydroxide/sodium carbonate; and j). Add saline to make up the volume.